Method of producing and processing diamines from an engineered microorganism

ABSTRACT

Provided is a method of producing and isolating a diamine produced by microbial fermentation that minimizes undesirable salt formation to provide a lower cost process.

PRIORITY CLAIM

-   -   This application claims priority from International Application        No. PCT/US2015/067478 filed Dec. 22, 2015, which in turn claims        the benefit of U.S. Provisional Patent Application Ser. No.        62/211,315 filed Aug. 28, 2015, U.S. Provisional Patent        Application Ser. No. 62/193,693 filed Jul. 17, 2015, and U.S.        Provisional patent Application Ser. No. 62/096,309 filed Dec.        23, 2014, the disclosure of each application incorporated herein        by reference.

The present invention generally provides a process to produce, isolate,and purify a diamine, including hexamethylenediamine (HMD), cadaverine,putrescine, ethylenediamine and heptamethylenediamine. The inventionmore particularly relates to a method for culturing a microorganismproducing the diamine, e.g. HMD, to a method of isolating the diaminefrom diamine-containing cultures or cultured media. Such diamineproducts are used to make diamine-containing polymers, includingpolyamides.

BACKGROUND

Hexamethylenediamine also referred to as 1,6-diaminohexane or1,6-hexanediamine (abbreviated as HMD or HMDA) has the chemical formulaH₂N(CH₂)₆NH₂. HMD is an important raw material in the chemical industry.HMD is used, for example, in the preparation of polyamides, polyureas orpolyurethanes and copolymers of these materials. Cadaverine, alsoreferred to as 1,5-diaminopentane, is used as a monomer for polyamineproduction. Putrescine, also referred to as 1,4-diaminobutane, is usedas a monomer for polyamine production. Heptamethylenediamine, alsoreferred to as 1,7-diaminoheptane, is used as a monomer for polyamineproduction. Ethylenediamine is used as a monomer for polyamineproduction as well as a precursor to other chemicals. Engineeredmicroorganisms for fermentative production of these compounds and otherdiamines or their immediate precursors have been reported. Typically,processes for their fermentation and isolation require acids and basesthat generate salt by-products.

SUMMARY OF THE INVENTION

An embodiment of the present invention utilizes carbon dioxide, addedexternally or produced metabolically, during a culture or fermentationprocess to produce a diamine species, at least one or more of diaminecarbonate, diamine bicarbonate, and/or diamine bis-bicarbonate(collectively referred to herein as “Carbonates”) and, optionallydiamine carbamate or diamine biscarbamate (collectively referred toherein as “Carbamates”). When the Carbonates and/or Carbamates areformed, the diamine species are neutralized and the fermentation pH iscontrolled. A carbon source for growth of the microorganism and itsproduction of the diamine is provided, as described below. Optionallythe carbon dioxide (or carbonate, bicarbonate) is both the carbon sourcefor the microorganism (via CO₂ fixation) and the compound forneutralizing the diamine. The diamine may be, for example,hexamethylenediamine (HMD), dimethylenediamine, trimethylenediamine,cadaverine, putrescine or heptamethylenediamine (diamines having two toseven carbon atoms (C2-C7), C3-C7, preferably C4-C7 or even C4 to C12 orC2-C12). Accordingly, the diamine species are, in the case of HMD forexample, HMD carbonate, HMD bicarbonate, HMD bis-bicarbonate. Thecarbamate and biscarbamate are, in the case of HMD for example, HMDcarbamate and HMD biscarbamate. The chemical formulas for the HMDspecies are shown below:

Free base H₂N—(CH₂)₆—NH₂ Bicarbonate [H₂N—(CH₂)₆—NH₃]⁺[HCO₃]⁻ Carbonate[H₃N—(CH₂)₆—NH₃]²⁺[CO₃]²⁻ Bis-bicarbonate [H₃N—(CH₂)₆−NH₃]²⁺[HCO₃]₂ ²⁻Carbamate H₂N−(CH₂)₆—NH—CO₂H Biscarbamate HO₂C—HN—(CH₂)₆—NH—CO₂H

In one embodiment, the present invention provides improved isolation ofCarbonates and/or Carbamates from culture or fermentation medium,solutions or broths, including Carbonates and Carbamates of HMD,cadaverine, putrescine and heptamethylenediamine. In another embodiment,the Carbonates and Carbamates are treated to release carbon dioxide anddiamine free base (e.g. HMD free base, cadaverine free base, putrescinefree base, heptamethylenediamine free base), and then the diamine freebase may be extracted with a suitable organic solvent. HMD-Carbonatesand -Carbamates produced during simulated fermentation conditions, suchas HMD carbonate, bicarbonate, bis-bicarbonate and carbamate andbiscarbamate, were found to release CO₂ or other fragments and togenerate HMD free base which was then solvent extracted. If necessary,the diamine free base enriched fraction is subject to furtherpurification processes.

In another embodiment, the invention provides a process for diamine (DA)production comprising the steps of:

-   -   a) culturing a genetically engineered microorganism in medium        under suitable conditions and for a sufficient period of time to        form one or more of DA carbonate, DA bicarbonate, DA        bis-bicarbonate (Carbonates) and/or DA carbamate or DA        biscarbamate (Carbamates) in the cultured medium wherein carbon        dioxide, carbonate, bicarbonate or carbonic acid predominantly        control pH of the medium as a cultured medium;    -   b) converting the DA carbonate, DA bicarbonate, DA        bis-bicarbonate, DA carbamate or DA biscarbamate into HMD free        base and carbon dioxide; and;    -   c) isolating the DA free base.

In another embodiment, the invention provides a process for diamine (DA)production comprising the steps of:

-   -   a) culturing a genetically engineered microorganism in medium        under suitable conditions and for a sufficient period of time to        form one or more of DA carbonate, DA bicarbonate, DA        bis-bicarbonate, DA carbamate or DA biscarbamate wherein        dissolved inorganic carbon percent (DIC) is determined by the        formula:        DIC/TDCA×100;        -   wherein the DIC % is greater than or equal to 40% and            wherein TDCA is the Total Dissolved Counter Anions and is            the sum of DIC and other anions;    -   b) converting the DA carbonate, DA bicarbonate, DA        bis-bicarbonate, DA carbamate or DA biscarbamate into DA free        base and carbon dioxide; and    -   c) isolating the DA free base.

In another embodiment, the invention provides a process for diamine (DA)production comprising the steps of:

-   -   a) culturing a genetically engineered microorganism in medium        comprising under suitable conditions and for a sufficient period        of time to produced DA and form one or more of DA carbonate, DA        bicarbonate, DA bis-bicarbonate (DA Carbonates) and/or DA        carbamate or DA biscarbamate (DA Carbamates) in the medium,        wherein at least 40% of Carbonates or Carbamates in the medium        comprises one or more of DA carbonate, DA bicarbonate, DA        bis-bicarbonate, DA carbamate or DA biscarbamate;    -   b) converting the Carbonates or Carbamates into DA free base and        carbon dioxide; and    -   c) isolating the DA free base.

In some embodiments, an enzyme carbonic anhydrase (CA) may be added tothe fermentation broth to catalyze or increase the formation of diamineCarbonates and/or diamine Carbamates (e.g. HMDA Carbonates) byincreasing the amount or rate of gaseous CO2 converted to soluble ion,thus providing a greater amount or availability of soluble ion availableto the diamine or HMD. The CA can also be used when the diaminecomprises C2 to C7 methylene segments, C2 to C12 methylene segments orC4 to C7 methylene segments, for example can be hexamethylenediamine(HMD), cadaverine, putrescine, ethylenediamine or heptamethylenediamine,to increase formation of the diamine Carbonates, including a carbonate,bicarbonate or bis-bicarbonate, and the diamine Carbamates, including acarbamate or biscarbamate, or any mixture thereof. In some embodiments,the carbonic anhydrase is used to form one of more HMD carbonates, HMDbicarbonate, HMD bis-bicarbonate, HMD carbamate or HMD biscarbamate.Carbonic anhydrase is a reversible enzyme and therefore in otherembodiments is used to catalyze the conversion of DA Carbonates or DACarbamates into DA free base and carbon dioxide.

In some embodiments, the carbonic anhydrase is present in sufficientamount to (a) enhance the formation of a DA Carbonates or DA Carbamatesby converting carbon dioxide to a bicarbonate and/or carbonate ions, (b)enhance the release of carbon dioxide from a solution of DA Carbonatesor DA Carbamates by converting a bicarbonate and/or carbonate ions tocarbon dioxide, or (c) both (a) and (b).

The CA may be added exogenously or may be produced by a geneticallyengineered microorganism. In some embodiments, the CA is part of amicroorganism that expresses a DA synthesis pathway such as HMDsynthesis pathway. In other embodiments, the CA is introduced as anengineered microorganism that has the ability to produce CA.

The CA or variant is expressed at sufficient amount to enhance eitherthe desired conversion of CO2 to ion or ion to CO2 or both, which can becompared to the conversion or conversions in the absence of the CA orvariant. An amount of CA or variant protein will depend on its carbonicanhydrase activity and desired enhancement. A typical amount can be inthe range of at least 0.001 g per liter to at least 5 gram per liter,and from at least 0.01 g/liter, 0.05 g/I, 0.1 g/liter, 0.2 g/liter, 0.5g/liter or 1 g/liter to at least 5 g/liter, for example 0.05 to 0.2g/liter.

Alternative embodiments are processes wherein at least 50% of Carbonatesand/or Carbamates in the medium comprises one or more of DA carbonate,DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA biscarbamate(e.g. HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDAcarbamate or HMDA biscarbamate), wherein at least 60% of Carbonatesand/or Carbamates in the medium comprises one or more of DA carbonate,DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA biscarbamate(e.g. HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDAcarbamate or HMDA biscarbamate), wherein at least 70% of Carbonatesand/or Carbamates in the medium comprises one or more of DA carbonate,DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA biscarbamate(e.g. HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDAcarbamate or HMDA biscarbamate), wherein at least 80% of Carbonatesand/or Carbamates in the medium comprises one or more of DA carbonate,DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA biscarbamate(e.g. HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDAcarbamate or HMDA biscarbamate), wherein at least 90% of Carbonatesand/or Carbamates in the medium comprises one or more of DA carbonate,DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA biscarbamate(e.g. HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDAcarbamate or HMDA biscarbamate), or wherein at least 99.9% of Carbonatesand/or Carbamates in the medium comprises one or more of DA carbonate,DA bicarbonate, DA bis-bicarbonate, DA carbamate or DA biscarbamate(e.g. HMDA carbonate, HMDA bicarbonate, HMDA bis-bicarbonate, HMDAcarbamate or HMDA biscarbamate). In some embodiments, the Carbonates arethe predominate diamine species and can include at least 50% of the DAspecies and up to at least 90%.

In some embodiments, the genetically engineered microorganism furtherforms one or more of carbon dioxide, carbonate, bicarbonate or carbonicacid. The genetically engineered microorganism formed carbon dioxide,carbonate, bicarbonate or carbonic acid may comprise stoichiometriccarbon dioxide from Carbonate and/or Carbamate formation, or thegenetically engineered microorganism formed carbon dioxide, carbonate,bicarbonate or carbonic acid may comprise respiration carbon dioxide orby-product carbon dioxide. In certain embodiments, the respirationcarbon dioxide is formed from at least one pathway selected from, forexample, via the completion of the TCA cycle, via the glyoxylate shunt,via the pentose phosphate pathway (e.g. gnd (6-phosphogluconatedehydrogenase that converts 6-phosphogluconate to ribuloase-5-phosphateand CO₂)), or via the Entner Duodoroff pathway. In other embodiments,the by-product carbon dioxide is associated with the formation ofby-products that include acetate, ethanol, succinate, 3-oxoadipate, and3-hydroxyadipate.

In some embodiments, a genetically engineered microorganism thatcomprises a diamine synthesis pathway, and optionally produces CO2, asdescribed herein, can further comprise a CA enzyme, particularly wherethe microorgansims is engineered to have a nucleic acid sequence capableof expressing CA. Accordingly, engineered microorganisms comprising asynthetic pathway to produce a diamine that comprises C2 to C7 methylenesegments, C2 to C12 methylene segments or C4 to C7 methylene segments,for example where the diamine is hexamethylenediamine (HMD), cadaverine,putrescine, ethylenediamine or heptamethylenediamine, can furthercomprise a CA enzyme, particularly where the microorgansims isengineered to have a nucleic acid sequence capable of expressing CA. Insome embodiments, a genetically engineered microorganism comprises ahexamethylenediamine synthesis pathway and sequences capable ofexpressing CA. In other embodiments, the genetically engineeredmicroorganism comprises sequences capable of expressing CA. The CA canbe native or genetically engineered, such as to increase activity orstability including thermal stability and alkaline pH stability.Preferably the alkaline pH is about pH 8-13, pH 8.5 to 13, pH 9-13, pH10-13, pH 8-12, pH 8.5-12, pH 9-12, pH 8-11, pH 8.5-11, pH 9-11, pH10-11 and pH 10-12.

In some embodiments, the genetically engineered microorganism formscarbon dioxide and hexamethylenediamine in a ratio of about 0.05 to 1 toabout 7 to 1. In other embodiments, the genetically engineeredmicroorganism forms carbon dioxide and hexamethylenediamine in a ratioof about 0.05 to 1 to about 5 to 1, in a ratio of about 0.05 to 1 toabout 3.5 to 1, in a ratio of about 0.05 to 1 to about 3 to 1, in aratio of about 0.05 to 1 to about 2 to 1, in a ratio of about 0.05 to 1to about 1.5 to 1, in a ratio of about 0.05 to 1 to about 1 to 1, or ina ratio of about 0.2 to 1 to about 3 to 1.

In some embodiments, the genetically engineered microorganism comprisesa HMD synthesis pathway with at least one exogenous nucleic acidencoding at least one enzyme of the HMD synthesis pathway expressed in asufficient amount to produce at least one HMD Carbonates and/orCarbamates compound. In still other embodiments, the geneticallyengineered microorganism comprises a HMD synthesis pathway with at leasttwo, three, four, five, six, seven, eight, nine, ten, or elevenexogenous nucleic acids encoding at least two, three, four, five, six,seven, eight, nine, ten or eleven enzymes of the HMD synthesis pathwayexpressed in a sufficient amount to produce at least one HMD Carbonatesand/or Carbamates compound.

In some embodiments, the HMD synthesis pathway comprises an intermediatecompound selected from the group consisting of 3-oxoadipyl-CoA, adipatesemialdehyde, 6-aminocaproate (6-ACA), 6-ACA semialdehyde,2-aminopimelate, 3,6-dihydroxyhexanoyl-CoA and homolysine.

In some embodiments, the HMD synthesis pathway comprises an enzymeselected from the group consisting of 3-oxoadipyl-CoA thiolase, 6-ACAtransaminase or dehydrogenase, 6-aminocaproyl-CoA reductase, 6-ACAreductase, adipyl-CoA reductase, adipate reductase, 6-hydroxy3-oxohexanoyl-CoA dehydrogenase, 2-aminopimelate decarboxylase, andhomolysine decarboxylase.

In some embodiments, the HMD synthesis pathway comprises an enzyme andsubstrate-product pair selected from the group consisting of3-oxoadipyl-CoA thiolase that acts on succinyl-CoA and acetyl-CoA tomake 3-oxoadipyl-CoA, 6-ACA transaminase that acts on adipyl-CoA to form6-ACA, 6-aminocaproyl-CoA reductase that acts on 6-aminocaproayl-CoA toform 6-ACA semialdehyde, 6-ACA reductase that acts on 6-ACA and convertsit directly to 6-ACA semialdehyde, adipyl-CoA reductase that acts onadipyl-CoA to form adipate semialdehyde, adipate reductase that acts onadipate and converts it directly to adipate semialdehyde, 6-hydroxy3-oxohexanoyl-CoA dehydrogenase that reduces 6-hydroxy 3-oxohexanoyl-CoAto form 3,6-dihydroxy hexanoyl-CoA, 2-aminopimelate decarboxylase thatdecarboxylates 2-aminopimelate to form 6-ACA, and homolysinedecarboxylase that decarboxylates homolysine to form HMD.

In some embodiments, the HMD synthesis pathway is selected from thegroup of pathways (a) to (m):

-   -   (a) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase,        3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA        reductase, adipyl-CoA reductase, 6-ACA transaminase or        dehydrogenase, 6-ACA transferase or synthetase and 6-ACA-CoA        reductase, or 6-ACA reductase, HMDA transaminase or        dehydrogenase;    -   (b) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase,        3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA        reductase, adipyl-CoA reductase, 6-ACA transaminase or        dehydrogenase, 6-ACA reductase, HMDA transaminase or        dehydrogenase;    -   (c) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase,        3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA        reductase, adipyl-CoA transferase, hydrolase or transferase,        adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA        transferase or synthetase, 6-ACA-CoA reductase, HMDA        transaminase or dehydrogenase;    -   (d) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase,        3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA        reductase, adipyl-CoA transferase, hydrolase or transferase,        adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA        reductase, HMDA transaminase or dehydrogenase;    -   (e) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase,        3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase,        adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA        transferase or synthetase, 6-ACA-CoA reductase, HMDA        transaminase or dehydrogenase;    -   (f) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase,        3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase,        adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA        reductase, HMDA transaminase or dehydrogenase;    -   (g) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase,        3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase,        adipyl-CoA transferase, hydrolase or transferase, adipyl-CoA        reductase, 6-ACA transaminase or dehydrogenase, 6-ACA        transferase or synthetase, 6-ACA-CoA reductase, HMDA        transaminase or dehydrogenase;    -   (h) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase,        3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase,        adipyl-CoA transferase, hydrolase or transferase, adipyl-CoA        reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase,        HMDA transaminase or dehydrogenase;    -   (i) an 4-hydroxy-2-oxoheptane-I,7-dioate (HODH aldolase); an        2-oxohept-4-ene-1,7-dioate (OHED) hydratase; an OHED        formate-lyase and a pyruvate formate-lyase activating enzyme or        OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an        adipyl-CoA dehydrogenase; or an adipate semialdehyde        aminotransferase or an adipate semialdehyde oxidoreductase        (aminating);    -   (j) a β-ketothiolase or an acetyl-CoA carboxylase and an        acetoacetyl-CoA synthase, a 3-hydroxyacyl-CoA dehydrogenase or a        3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, and a        trans-2-enoyl-CoA reductase for producing hexanoyl-CoA, one or        more of a thioesterase, an aldehyde dehydrogenase, or a butanal        dehydrogenase, said host producing hexanal or hexanoates; one or        more of a monooxygenase, an alcohol dehydrogenase, an aldehyde        dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a        5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate        dehydrogenase, a 6-oxohexanoate dehydrogenase, or a        7-oxoheptanoate dehydrogenase, said host producing adipic acid        or adipate semialdehyde; one or more of a monooxygenase, a        transaminase, a 6-hydroxyhexanoate dehydrogenase, a        5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate        dehydrogenase, and an alcohol dehydrogenase, said host producing        6-aminohexanoate; one or more of a carboxylate reductase, a        w-transaminase, a deacetylase, a N-acetyl transferase, or an        alcohol dehydrogenase, said host producing hexamethylenediamine;    -   (k) acetyltransferase or thiolase to form        6-hydroxy-3-oxo-hexanoyl-CoA, 6-hydroxy-3-oxo-hexanoyl-CoA        dehydrogenase, 3,4-dihydroxyhexanoyl-CoA dehydratase,        6-hydroxy-2-hexenoyl-CoA reductase, 6-hydroxyhexanoyl-CoA        hydrolase to form 6-ACA, 6-hydroxycaproate dehydrogenase and        transaminase to form HMDA;    -   (l) homocitrate synthase, a homoaconitase and a homoisocitrate        dehydrogenase to form 2-ketopimelate, 2-keto decarboxylase        catalyzing the conversion of α-ketopimelate to adipate        semialdehyde, 2-aminotransferase catalyzes the conversion of        α-ketopimelate to 2-aminopimelate, 2-aminopimelate decarboxylase        to decarboxylate 2-aminopimelate and form 6-ACA, aldehyde        dehydrogenase catalyzes the conversion of 6-ACA to        6-aminohexanal and the aminotransferase catalyzes the conversion        of 6-aminohexanal to 6-hexamethylenediamine; and    -   (m) glutamyl-CoA transferase and/or ligase, beta-ketothiolase,        3-oxo-6-aminopimeloyl-CoA oxidoreductase,        3-hydroxy-6-aminopimeloyl-CoA dehydratase,        6-amino-7-carboxyhept-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA        reductase (aldehyde forming), 2-amino-7-oxoheptanoate        aminotransferase and/or aminating oxidoreductase, homolysine        decarboxylase, 6-aminopimeloyl-CoA hydrolase, transferase and/or        ligase, 2-aminopimelate decarboxylase.

In any of the embodiments in the alternative pathways set out above,suitable enzymes may be selected from the group consisting of3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase,3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase,adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase,3-oxoadipyl-CoA:acyl CoA transferase, 3-oxoadipate dehydrogenase,3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase,adipyl-CoA transferase, lygase or hydrolase, 6-ACA transferase orsynthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase,adipate reductase, 6-ACA transaminase or dehydrogenase, or 6-ACAreductase.

In some embodiments, the genetically engineered microorganism includethe genus Escherichia, Klebsiella; the order Aeromoriadales, familySuccinivibrionaceae, including the genus Anaerobiospirillum; the orderPasteurellales, family Pasteurellaceae, including the generaActinobacillus and Mannheimia; the order Rhizobiales, familyBradyrhizobiaceae, including the genus Rhizobium; the order Bacillales,family Bacillaceae, including the genus Bacillus; the orderActinomycetales, families Corynebacteriaceae and Streptomycetaceae,including the genus Corynebacterium and the genus Streptomyces,respectively; order Rhodospirillales, family Acetobacteraceae, includingthe genus Gluconobacter; the order Sphingomonadales, familySphingomonadaceae, including the genus Zymomonas; the orderLactobacillales, families Lactobacillaceae and Streptococcaceae,including the genus Lactobacillus and the genus Lactococcus,respectively; the order Clostridiales, family Clostridiaceae, genusClostridium; and the order Pseudomonadales, family Pseudomonadaceae,including the genus Pseudomonas, the genus Alkaliphilus,Methylobacterium, Methyloversatilis, Methylococcus, Methylocystis andHyphomicrobium the order Saccharomycetales, family Saccaromycetaceae,including the genera Saccharomyces, Kluyveromyces and Pichia; the orderSaccharomycetales, family Dipodascaceae, including the genus Yarrowia;the order Schizosaccharomycetales, family Schizosaccaromycetaceae,including the genus Schizosaccharomyces; the order Eurotiales, familyTrichocomaceae, including the genus Aspergillus; and the orderMucorales, family Mucoraceae, including the genus Rhizopus.

In other embodiments the genetically engineered microorganism includeEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Corynebacterium glutamicum,Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis,Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida,Bacillis pseudofirmus, Bacillus halodurans, Bacillus alcalophilus,Clostridium paradoxum, Saccharomyces cerevisiae, Schizosaccharomycespombe, Hansenula polymorpha, Pichia methanolica, Candida boidinii,Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus,Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae,Yarrowia lipolytica, and Issatchenkia orientalis.

Some emodiments of alkaliphiles are: Bacillis pseudofirmus, Bacillushalodurans, Bacillus alcalophilus, Clostridium paradoxum, Arthrospiraplatensis, Bacillus clausii, Oceanobacillus iheyensis, Alkaliphilusmetaffiredigens, Alkaliphilus oremlandii, Bacillus selentireducens,Desulfovibrio alkaliphiles, Dethiobacter alkaliphiles, Thioalkalivibriosp., Natranaerobius thermophilus, Alkalilimnicola ehrlichii, andDesulfonatronospira thiodismutans.

In some embodiments, the culture medium fermentation may besubstantially free of a buffer, may be substantially free of inorganicor organic acid, substantially free of externally added inorganic ororganic acid or substantially free of DIC.

In some embodiments, the medium and/or cultured medium pH is controlledby carbon dioxide amount added to the culture medium, or alternatively,the cultured medium pH is controlled by the amount of carbon dioxideformed by the genetically engineered microorganism. In certainembodiments, the medium has a pH and/or the cultured medium iscontrolled to a pH of less than 11, less than 10, less than 9, or lessthan 8. In other embodiments, the medium has a pH and/or the culturedmedium is controlled to a pH of at least 2, at least 3, at least 4, atleast 5, at least 6, or at least 7. In still other embodiments, themedium has a pH and/or the cultured medium is controlled to a pH ofabout 6 to 9.5, a pH of about 6 to 9, a pH of about 6 to 8, a pH ofabout 7-9 or a pH of about 8-9.

In still other embodiments, the medium comprises a sugar carbon sourcefor the genetically engineered microorganism selected from the groupconsisting of sucrose, glucose, galactose, fructose, starch, mannose,isomaltose, xylose, pannose, maltose, arabinose, cellobiose and 3-, 4-,or 5-oligomers thereof, or the medium comprises an alcohol carbon sourcefor the genetically engineered microorganism selected from the groupconsisting of methanol, ethanol, glycerol, formate and fatty acids, orthe medium comprises a carbon source obtained from gas for thegenetically engineered microorganism selected from the group consistingof synthesis gas, waste gas, methane, CO, CO₂, and any mixture of CO orCO₂ with H₂.

In some embodiments, the Carbonates and/or Carbamates are converted tothe free base, e.g. hexamethylenediamine free base, by generating carbondioxide. In certain embodiments, the Carbonates and/or Carbamates areconverted to the free base by heat, the Carbonates and/or Carbamates areconverted to the free base by vacuum, the Carbonates and/or Carbamatesare converted to the free base by pressure, the Carbonates and/orCarbamates are converted to the free base by ion exchange, theCarbonates and/or Carbamates are converted to the free base by steamstripping, or the Carbonates and/or Carbamates are converted to the freebase by electrodialysis using a bipolar membrane. In still otherembodiments, the conversion to the free base is accelerated by orenhanced by the addition of a carbonic anhydrase enzyme. The carbonicanhydrase may also be used to accelerate or enhance the release to freebase when heat or other steps are used to convert the DA carbonatesand/or DA Carbamates to free DA and carbon dioxide.

In some embodiments the diamine free base (e.g. HMD) is isolated fromthe medium using an extraction solvent and the extracted diamine isseparated from the extraction solvent by distillation. In certainembodiments the extraction solvent is selected from the group consistingof alcohols, amines, ethers and ketones. Suitable extraction solventscomprise C4-C8 monohydric alcohols such as butanol, hexanal, 1-hexanol,isopentanol, or cyclohexanol, or alternatively toluene or ethyl ether ormixtures thereof. Alkanes are suitable solvents as demonstrated in theExamples, particularly for HMD free base. Alkanes, specifically hexane,were screened and subsequently tested due to extremely low watersolubility. Hexane extracted little if any water and provided reasonablerecovery of the available free base. Alkanes are therefore suitablesolvents for use in recovery of diamine free base. Suitable alkanesinclude C5-C12, linear or branched. In one embodiment, both the diamineto be extracted and the alkane selected as solvent will have the samenumber of carbon atoms. Heptane is another suitable alkane, especiallyfor HMD, which is further supported by the in silico modeling studybelow. Isomers of hexane and heptane are suitable. Hexane isomers are2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, and2,3-dimethylbutane. Heptane isomers are 2-methylhexane, 3-methylhexane,2,2-dimethylpentane, 2,3-dimethylpentane, 2,4-dimethylpentane,3,3-dimethylpentane, 3-ethylpentane and 2,2,3-trimethylbutane.

In some embodiments, the genetically engineered microorganism isEscherichia coli, Corynebacterium glutamicum, Bacillus subtilis,Pseudomonas putida, Bacillis pseudofirmus, Bacillus halodurans, Bacillusalcalophilus, Clostridium paradoxum, Saccharomyces cerevisiae. In otherembodiments, the genetically engineered microorganism is modified forimproved alkali tolerance.

In some embodiments, the DA, e.g. HMD, produced by the present inventioncomprises one or more of DA carbonate, DA bicarbonate, DAbis-bicarbonate or DA carbamate impurities.

In some embodiments, a polymer, e.g. a polyamide for example PA66,comprising the diamine, e.g. HMD, produced by the present processcomprises one or more of the DA carbonate, DA bicarbonate, DAbis-bicarbonate, DA carbamate or DA biscarbamate (e.g. HMD carbonate,HMD bicarbonate, HMD bis-bicarbonate, HMD carbamate or HMD biscarbamate)as impurities.

Another embodiment of the invention may be a genetically engineeredmicroorganism comprising a diamine synthesis pathway, e.g. ahexamethylenediamine synthesis pathway, with at least one exogenousnucleic acid encoding at least one enzyme of the diamine synthesispathway, e.g. HMD synthesis pathway, and at least one geneticmodification that enhances or increases CO₂ availability to increaseproduction of a diamine Carbonate and/or Carbamate, e.g. HMD Carbonateand/or Carbamate, compared to a genetically engineered microorganismabsent that genetic modification. In one embodiment, a geneticallyengineered microorganism comprising the diamine, e.g.hexamethylenediamine, synthesis pathway with at least one exogenousnucleic acid encoding at least one enzyme of the diamine, e.g. HMD,synthesis pathway, and at least one carbonic anhydrase enzyme is used toincrease production of the diamine, e.g. HMD, Carbonate and/or Carbamatecompared to a genetically engineered microorganism absent the CA enzyme.The CA-expressing microorganism can further comprise the at least onegenetic modification that increases CO₂ availability.

In any of the embodiments of the present invention, released carbondioxide, the extraction solvent and/or water may be recycled. In otherembodiments, the CA may be recycled.

In addition to the present process steps of culturing, converting andisolating described in the above embodiments, the present inventionsalso includes alternative and optional process steps. In someembodiments, the cultured medium or solution may be treated to removesolids and water during the process, either before isolating the DA freebase and/or before converting the Carbonates and/or Carbamates to the DAfree base. In other embodiments, the cultured medium may be treated toremove water, preferably before isolating the DA free based. In stillother embodiments, the DA free base may be directly distilled from thecultured medium or solution. In still other embodiments, the DA freebase be further treated and or purified after the extraction solvent isremoved by distillation. In still other embodiments, the water removalor reduction and the conversion of the Carbonates and/or Carbamates tothe DA free base, e.g. HMD free base) occur simultaneously and/orsequentially in the same unit operation. For example, the stripper unit(e.g. inert gas or steam) if present can be used to remove or reduceboth water and CO2 to generate the free base. For a further example, thewater evaporator unit if present can be used to remove or reduce bothwater and CO2 to generate the free base. The CA may be present duringfermentation for formation of the diamine Carbonate and/or Carbamate ormay be present during the step or steps for release of CO2 or may bepresent at either or both steps. In some embodiments, he CA may berecycled.

These alternative and/or optional process steps are described in detailbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart illustrating one embodiment of the invention ofpreparing a diamine.

FIG. 2 is a flow chart illustrating another embodiment of the inventionof preparing diamine.

FIG. 3 is a schematic flow chart illustrating another embodiment of theinvention of a fermentation process to prepare diamine.

FIG. 4 is a block diagram of steps in an embodiment for preparing andprocessing a diamine. The numbers in each box are as follows:

1: Product fermentation

2: Microbiol inactivation/Heat kill. May result in partial release ofcarbon dioxide and free base due to elevated temperature

3: Solids removal

4: Carbonate/Carbamate conversion to carbon dioxide and free base.Carbon dioxide is optionally recycled to fermentor.

5: Water removal. Water is optionally recycled to the productfermentation step. Carbon dioxide can also be recycled if water removalstep involves conditions that can release carbon dioxide and free base.

6: Solvent extraction. Aqueous raffinate is optionally recycled to theCarbonate/Carbamate conversion step.

7: Purification: Involves distillation which recycles organic solventback to box 6; could involve more distillation columns to purify HMD andother steps to remove color forming compounds, etc.

8: Purified HMD

9: Optional Sterilization where no carbon dioxide is released

10: Optional water removal. Water is optionally recycled to the productfermentation step. Carbon dioxide can also be recycled if water removalstep involves conditions that can release carbon dioxide and free base.

11: Optional direct purification from aqueous phase with or withoutrelease of carbon dioxide. This could involve distillation, ionexchange, electrodialysis, etc. Possible to recycle water and carbondioxide if it is produced in these steps

12: Alkalization (NaOH or CaOH) or other steps to remove Carbonates fromHMD (Ion exchange, electrodialysis, etc.). If CaOH is used a precipitatewill form.

FIG. 5 shows an exemplary pathway for synthesis of adipic acid fromglucose via cis,cis-muconic acid. Biosynthetic intermediates(abbreviations): D-erythrose 4-phosphate (E4P), phosphoenolpyruvic acid(PEP), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP),3-dehydroquinic acid (DHQ), 3-dehydroshikimic acid (DHS), protocatechuicacid (PCA). Enzymes (encoding genes) or reaction conditions: (a) DAHPsynthase (aroFFBR), (b) 3-dehydroquinate synthase (aroB), (c)3-dehydroquinate dehydratase (aroD), (d) DHS dehydratase (aroZ), (e)protocatechuate decarboxylase (aroY), (f) catechol 1,2-dioxygenase(catA), (g) 10% Pt/C, H.sub.2, 3400 kPa, 25.degree. C. Figure taken fromNiu et al., Biotechnol. Prog. 18:201-211 (2002).

FIG. 6 shows an exemplary pathway for adipate synthesis viaalpha-ketoadipate using alpha-ketoglutarate as a starting point.

FIG. 7 shows an exemplary pathway for synthesis of adipate using lysineas a starting point.

FIG. 8 shows an exemplary caprolactam synthesis pathway using adipyl-CoAas a starting point.

FIG. 9 shows exemplary adipate synthesis pathways usingalpha-ketoadipate as a starting point.

FIG. 10 shows exemplary pathways from succinyl-CoA and acetyl-CoA tohexamethylenediamine (HMDA) and caprolactam. Pathways for the productionof adipate, 6-aminocaproate, caprolactam, and hexamethylenediamine fromsuccinyl-CoA and acetyl-CoA are depicted. Abbreviations: A)3-oxoadipyl-CoA thiolase, B) 3-oxoadipyl-CoA reductase, C)3-hydroxyadipyl-CoA dehydratase, D) 5-carboxy-2-pentenoyl-CoA reductase,E) 3-oxoadipyl-CoA/acyl-CoA transferase, F) 3-oxoadipyl-CoA synthase, G)3-oxoadipyl-CoA hydrolase, H) 3-oxoadipate reductase, I)3-hydroxyadipate dehydratase, J) 5-carboxy-2-pentenoate reductase, K)adipyl-CoA/acyl-CoA transferase, L) adipyl-CoA synthase, M) adipyl-CoAhydrolase, N) adipyl-CoA reductase (aldehyde forming), O)6-aminocaproate transaminase, P) 6-aminocaproate dehydrogenase, Q)6-aminocaproyl-CoA/acyl-CoA transferase, R) 6-aminocaproyl-CoA synthase,S) amidohydrolase, T) spontaneous cyclization, U) 6-aminocaproyl-CoAreductase (aldehyde forming), V) HMDA transaminase, W) HMDAdehydrogenase.

FIG. 11 shows exemplary pathways from 4-aminobutyryl-CoA and acetyl-CoAto hexamethylenediamine and caprolactam. Pathways for the production of6-aminocaproate, caprolactam, and hexamethylenediamine from4-aminobutyryl-CoA and acetyl-CoA are depicted. Abbreviations: A)3-oxo-6-aminohexanoyl-CoA thiolase, B) 3-oxo-6-aminohexanoyl-CoAreductase, C) 3-hydroxy-6-aminohexanoyl-CoA dehydratase, D)6-aminohex-2-enoyl-CoA reductase, E) 3-oxo-6-aminohexanoyl-CoA/acyl-CoAtransferase, F) 3-oxo-6-aminohexanoyl-CoA synthase, G)3-oxo-6-aminohexanoyl-CoA hydrolase, H) 3-oxo-6-aminohexanoatereductase, I) 3-hydroxy-6-aminohexanoate dehydratase, J)6-aminohex-2-enoate reductase, K) 6-aminocaproyl-CoA/acyl-CoAtransferase, L) 6-aminocaproyl-CoA synthase, M) 6-aminocaproyl-CoAhydrolase, N) 6-aminocaproyl-CoA reductase (aldehyde forming), O) HMDAtransaminase, P) HMDA dehydrogenase, Q) spontaneous cyclization, R)amidohydrolase.

FIG. 12 shows pathways to 6-aminocaproate from pyruvate and succinicsemialdehyde. Enzymes are A) HODH aldolase, B) OHED hydratase, C) OHEDreductase, D) 2-OHD decarboxylase, E) adipate semialdehydeaminotransferase and/or adipate semialdehyde oxidoreductase (aminating),F) OHED decarboxylase, G) 6-OHE reductase, H) 2-OHD aminotransferaseand/or 2-OHD oxidoreductase (aminating), I) 2-AHD decarboxylase, J) OHEDaminotransferase and/or OHED oxidoreductase (aminating), K) 2-AHEreductase, L) HODH formate-lyase and/or HODH dehydrogenase, M)3-hydroxyadipyl-CoA dehydratase, N) 2,3-dehydroadipyl-CoA reductase, O)adipyl-CoA dehydrogenase, P) OHED formate-lyase and/or OHEDdehydrogenase, Q) 2-OHD formate-lyase and/or 2-OHD dehydrogenase.Abbreviations are: HODH=4-hydroxy-2-oxoheptane-1,7-dioate,OHED=2-oxohept-4-ene-1,7-dioate, 2-OHD=2-oxoheptane-1,7-dioate,2-AHE=2-aminohept-4-ene-1,7-dioate, 2-AHD=2-aminoheptane-1,7-dioate, and6-OHE=6-oxohex-4-enoate.

FIG. 13 shows pathways to hexamethylenediamine from 6-aminocapropate.Enzymes are A) 6-aminocaproate kinase, B) 6-AHOP oxidoreductase, C)6-aminocaproic semialdehyde aminotransferase and/or 6-aminocaproicsemialdehyde oxidoreductase (aminating), D) 6-aminocaproateN-acetyltransferase, E) 6-acetamidohexanoate kinase, F) 6-AAHOPoxidoreductase, G) 6-acetamidohexanal aminotransferase and/or6-acetamidohexanal oxidoreductase (aminating), H) 6-acetamidohexanamineN-acetyltransferase and/or 6-acetamidohexanamine hydrolase (amide), I)6-acetamidohexanoate CoA transferase and/or 6-acetamidohexanoate CoAligase, J) 6-acetamidohexanoyl-CoA oxidoreductase, K) 6-AAHOPacyltransferase, L) 6-AHOP acyltransferase, M) 6-aminocaproate CoAtransferase and/or 6-aminocaproate CoA ligase, N) 6-aminocaproyl-CoAoxidoreductase. Abbreviations are:6-AAHOP=[(6-acetamidohexanoyl)oxy]phosphonate and6-AHOP=[(6-aminohexanoyl)oxy]phosphonate.

FIG. 14 shows: A) the acetyl-CoA cycle of arginine biosynthesis.Reactions (1) and (2) are catalyzed by ornithine acetyltransferase withacetylgiutamate synthase and ornithine acyltransferase functionality.Reaction 3 is a lumped reaction catalyzed by acetylglutamate kinase,N-acetylglutamylphosphate reductase, and acetylornithineaminotransferase; B) the acetyl-CoA cycle of HMDA biosynthesis.Reactions (1) and (2) are catalyzed by HMDA acetyltransferase. Reaction(3) is a lumped reaction that includes all pathways to6-acetamidohexanamine from 6-acetamidohexanoate shown in FIG. 13.

FIG. 15 shows exemplary pathways from glutamate to hexamethylenediamine(HMDA) and 6-aminocaproate. The enzymes are designated as follows: A)glutamyl-CoA transferase and/or ligase, B) beta-ketothiolase, C)3-oxo-6-aminopimeloyl-CoA oxidoreductase, D)3-hydroxy-6-aminopimeloyl-CoA dehydratase, E)6-amino-7-carboxyhept-2-enoyl-CoA reductase, F) 6-aminopimeloyl-CoAreductase (aldehyde forming), G) 2-amino-7-oxoheptanoateaminotransferase and/or aminating oxidoreductase, H) homolysinedecarboxylase, I) 6-aminopimeloyl-CoA hydrolase, transferase and/orligase, J) 2-aminopimelate decarboxylase.

FIG. 16 shows exemplary pathways from glutaryl-CoA tohexamethylenediamine (HMDA) and 6-aminocaproate. The enzymes aredesignated as follows: A) glutaryl-CoA beta-ketothiolase, B)3-oxopimeloyl-CoA hydrolase, transferase and/or ligase, C) 3-oxopimelatereductase, D) 3-oxo-1-carboxyheptanal 7-aminotransferase and/or7-aminating oxidoreductase, E) 3-oxo-7-aminoheptanoate3-aminotransferase and/or 3-aminating oxidoreductase, F) 3-oxopimelatekinase, G) 5-oxopimeloylphosphonate reductase, H) 3-oxopimelate CoAtransferase and/or ligase, I) 5-oxopimeloyl-CoA reductase (aldehydeforming), J) 3-oxopimelate 3-aminotransferase and/or 3-aminatingoxidoreductase, K) 3-aminopimelate CoA transferase and/or ligase, L)5-aminopimeloyl-CoA reductase (aldehyde forming), M) 3-aminopimelatekinase, N) 5-aminopimeloylphosphonate reductase, O) 3-aminopimelatereductase, P) 3-amino-7-oxoheptanoate 2,3-aminomutase, Q)2-amino-7-oxoheptanoate 7-aminotransferase and/or aminatingoxidoreductase, R) 3,7-diaminoheptanoate 2,3-aminomutase, S) homolysinedecarboxylase, T) 3-aminopimelate 2,3-aminomutase, U) 2-aminopimelatekinase, V) 2-aminopimelate CoA transferase and/or ligase, W)2-aminopimelate reductase, X) 6-aminopimeloylphosphonate reductase, Y)6-aminopimeloyl-CoA reductase (aldehyde forming), Z)3-amino-7-oxoheptanoate 7-aminotransferase and/or 7-aminatingoxidoreductase, AA) 2-aminopimelate decarboxylase and AB)3-oxo-1-carboxyheptanal 3-aminotransferase and/or 3-aminatingoxidoreductase.

FIG. 17 shows an exemplary pathway from pyruvate and 4-aminobutanal tohexamethylenediamine (HMDA). The enzymes are designated as follows: A)2-oxo-4-hydroxy-7-aminoheptanoate aldolase, B)2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, C)2-oxo-7-aminohept-3-enoate reductase, D) 2-oxo-7-aminoheptanoateaminotransferase and/or aminating oxidoreductase, E) homolysinedecarboxylase, F) 2-oxo-7-aminoheptanoate decarboxylase, G)6-aminohexanal aminotransferase and/or 6-aminohexanal aminatingoxidoreductase.

FIG. 18 shows an exemplary pathway from homolysine to 6-aminocaproate.Step A is catalyzed by homolysine 2-monooxygenase. Step B is hydrolysis,catalyzed by dilute acid or base.

FIG. 19 shows exemplary pathways from 6-aminocaproate tohexamethylenediamine. This figure depicts additional pathways further tothose presented in FIG. 13. The enzymes are designated as follows: A)6-aminocaproate kinase, B) 6-AHOP oxidoreductase, C) 6-aminocaproicsemialdehyde aminotransferase and/or 6-aminocaproic semialdehydeoxidoreductase (aminating), D) 6-aminocaproate N-acetyltransferase, E)6-acetamidohexanoate kinase, F) 6-AAHOP oxidoreductase, G)6-acetamidohexanal aminotransferase and/or 6-acetamidohexanaloxidoreductase (aminating), H) 6-acetamidohexanamine N-acetyltransferaseand/or 6-acetamidohexanamine hydrolase (amide), I) 6-acetamidohexanoateCoA transferase and/or 6-acetamidohexanoate CoA ligase, J)6-acetamidohexanoyl-CoA oxidoreductase, K) 6-AAHOP acyltransferase, L)6-AHOP acyltransferase, M) 6-aminocaproate CoA transferase and/or6-aminocaproate CoA ligase, N) 6-aminocaproyl-CoA oxidoreductase, O)6-aminocaproate reductase and P) 6-acetamidohexanoate reductase.Abbreviations are: 6-AAHOP=[(6-acetamidohexanoyl)oxy]phosphonate and6-AHOP=[(6-aminohexanoyl)oxy]phosphonate.

FIG. 20 shows exemplary pathways from succinyl-CoA and acetyl-CoA tohexamethylenediamine (HMDA), caprolactam or levulinic acid. Pathways forthe production of adipate, 6-aminocaproate, caprolactam,hexamethylenediamine and levulinic acid from succinyl-CoA and acetyl-CoAare depicted. This figure depicts additional pathways further to thosepresented in FIG. 10. The enzymes are designated as follows: A)3-oxoadipyl-CoA thiolase, B) 3-oxoadipyl-CoA reductase, C)3-hydroxyadipyl-CoA dehydratase, D) 5-carboxy-2-pentenoyl-CoA reductase,E) 3-oxoadipyl-CoA/acyl-CoA transferase, F) 3-oxoadipyl-CoA synthase, G)3-oxoadipyl-CoA hydrolase, H) 3-oxoadipate reductase, I)3-hydroxyadipate dehydratase, J) 5-carboxy-2-pentenoate reductase, K)adipyl-CoA/acyl-CoA transferase, L) adipyl-CoA synthase, M) adipyl-CoAhydrolase, N) adipyl-CoA reductase (aldehyde forming), O)6-aminocaproate transaminase, P) 6-aminocaproate dehydrogenase, Q)6-aminocaproyl-CoA/acyl-CoA transferase, R) 6-aminocaproyl-CoA synthase,S) amidohydrolase, T) spontaneous cyclization, U) 6-aminocaproyl-CoAreductase (aldehyde forming), V) HMDA transaminase, W) HMDAdehydrogenase, X) adipate reductase, Y) adipate kinase, Z)adipylphosphate reductase, and AA) 3-oxoadipate decarboxylase.

FIG. 21 shows exemplary pathways from 2-amino-7-oxosubarate tohexamethylenediamine (HMDA) and 6-aminocaproate. The enzymes aredesignated as follows: A) 2-amino-7-oxosubarate keto-acid decarboxylase,B) 2-amino-7-oxoheptanoate decarboxylase, C) 6-aminohexanal aminatingoxidoreductase and/or 6-aminohexanal aminotransferase, D)2-amino-7-oxoheptanoate oxidoreductase, E) 2-aminopimelatedecarboxylase, F) 6-aminohexanal oxidoreductase, G)2-amino-7-oxoheptanoate decarboxylase, H) homolysine decarboxylase, I)2-amino-7-oxosubarate amino acid decarboxylase, J)2-oxo-7-aminoheptanoate aminating oxidoreductase and/or2-oxo-7-aminoheptanoate aminotransferase, K) 2-amino-7-oxosubarateaminating oxidoreductase and/or 2-amino-7-oxosubarate aminotransferase,L) 2,7-diaminosubarate decarboxylase and M) 2-amino-7-oxoheptanoateaminating oxidoreductase and/or 2-amino-7-oxoheptanoateaminotransferase.

FIG. 22 shows an exemplary pathway from glutamate-5-semialdehyde to2-amino-7-oxosubarate. The enzymes are designated as follows: A)2-amino-5-hydroxy-7-oxosubarate aldolase, B)2-amino-5-hydroxy-7-oxosubarate dehydratase, C)2-amino-5-ene-7-oxosubarate reductase.

FIG. 23 shows an exemplary pathway for adipate degradation in theperoxisome of Penicillium chrysogenum.

FIG. 24 shows an exemplary pathway for adipate formation via a reversedegradation pathway. Several options are provided for the finalconversion of adipyl-CoA to adipate.

FIG. 25 shows an exemplary pathway for adipate formation via the3-oxoadipate pathway.

FIG. 26 shows the similar enzyme chemistries of the last three steps ofthe 3-oxoadipate pathway for adipate synthesis and the reductive TCAcycle.

FIG. 27 shows a graphical representation of pH as function of timebubbling in CO₂ over 60 minutes.

FIG. 28 shows a graphical representation of pH as a function of timebubbling in CO₂ for the first 10 minutes.

FIG. 29 shows a graphical representation of pH as a function of processconditions shown in Table 1-1.

FIG. 30 shows a graphical representation of pH and HMD concentrationinside a fermentor as a function of time.

FIG. 31 shows a graphical representation of concentration of HMD speciesas a function of pH.

FIG. 32 shows a graphical representation of carbonate species as afunction of pH.

FIG. 33 shows a graphical representation of alkanes as solvents for HMDrecovery.

FIG. 34 shows a graphical representation of the effect of DIC on HMDextraction efficiency into hexane.

FIG. 35 shows a graphical representation of steam and electricity usagesas a function of total water removed when no stripping column ispresent.

FIG. 36 shows a graphical representation of steam usage in a strippingcolumn when no evaporator used.

DETAILED DESCRIPTION

Disclosed is a process for the production and isolation of a diamine. Asreferred here, “diamines” include C2 to C7 methylene segments such ashexamethylenediamine (HMD), cadaverine, putrescine, ethylenediamine andheptamethylenediamine, for example. The diamines can have carbon contentof C2-C7, C3-C7, preferably C4-C7 or even C4-C12 or C2-C12. It should beunderstood that for ease of reading, HMD may be described in detail butthe disclosed process is applicable to any of the diamines such ascadaverine, putrescine, ethylenediamine and heptamethylenediamine.

Hexamethylenediamine also referred to as 1,6-diaminohexane or1,6-hexanediamine (abbreviated as HMD) has the chemical formulaH2N(CH2)6NH2. HMD is an important raw material in the chemical industry.HMD is used, for example, in the preparation of polyamides, polyureas orpolyurethanes and copolymers of these materials.

Cadaverine, also referred to as 1,5-diaminopentane is used as a monomerfor polyamine production. Engineered microorganisms suitable forfermentative production of cadaverine have been reported. For example, amethod to produce and recover a bio-based amine (e.g. cadaverine) isreported in U.S. Pat. No. 8,906,653 and in the literature by Kind et al.“From zero to hero—Production of bio-based nylon from renewableresources using engineered Corynebacterium glutamicum,” (MetabolicEngineering, 25 (2014) pp. 113-123) and Kind et al. “Systems-widemetabolic pathway engineering in Corynebacterium glutamicum forbio-based production of diaminopentane,” (Metabolic Engineering 12(2010)341-351). These reported processes involve active neutralizationof a fermentation broth or cultured medium with an inorganic acid (e.g.sulfuric acid). After fermentation, the cultured medium or broth isalkalized with a strong base to deprotonate the amine, which is thenextracted with an organic solvent and subsequently distilled. In theseprocesses, copious amounts of unwanted salt by-products are producedwith the amine.

In another method, lysine carbonate prepared in vitro is enzymaticallydecarboxylated to produce cadaverine carbonate with addition of adicarboxylic acid salt to maintain suitable pH for the decarboxylationreaction, followed by concentration to generate cadaverine andcadaverine-dicarboxylic acid salt, as reported in International PatentApplication Publication No. WO 2006/123778. In still another method,lysine carbonate prepared in vitro is enzymatically decarboxylated toproduce cadaverine carbonate which is thermally treated and thendistilled to provide cadaverine as reported in International PatentApplication Publication No. WO 2010/002000.

Putrescine, also referred to as 1,4-diaminobutane is used as a monomerfor polyamine production. Engineered microorganisms suitable forfermentative production of putrescine have been reported. See, forexample, Schneider et al. “Improving putrescine production byCorynebacterium glutamicum by fine-tuning ornithine transcarbamoylaseactivity using a plasmid addition system,” (Appl Microbiol Biotechnol.2012; 95(1):169-78); and U.S. Patent Application Publication No.20140004577A1 “Microorganisms for producing putrescine and method forproducing putrescine using same.”

Heptamethylenediamine, also referred to as 1,7-diaminoheptane, is usedas a monomer for polyamine production. Engineered microorganismssuitable for fermentative production of putrescine have been reported.See, for example International Patent Application Publication No.WO2014105790A2, “Methods of producing 7-carbon chemicals via c1 carbonchain elongation associated with coenzyme b synthesis.”

Ethylenediamine is used as a monomer for polyamine production as well asa precursor to other chemicals. Engineered microorganisms forfermentative production of ethylenediamine have been reported. See forexample International Patent Application Publication No. WO2014049382A2,“Ethylenediamine fermentative production by a recombinantmicroorganism.”

Hexamethylenediamine also referred to as 1,6-diaminohexane or1,6-hexanediamine (abbreviated as HMD) has the chemical formulaH₂N(CH₂)₆NH₂. HMD is an important raw material in the chemical industry.HMD is used, for example, in the preparation of polyamides, polyureas orpolyurethanes and copolymers of these materials. Cadaverine, alsoreferred to as 1,5-diaminopentane is used as a monomer for polyamineproduction. Putrescine, also referred to as 1,4-diaminobutane is used asa monomer for polyamine production. Heptamethylenediamine, also referredto as 1,7-diaminoheptane, is used as a monomer for polyamine production.Ethylenediamine is used as a monomer for polyamine production as well asa precursor to other chemicals. Engineered microorganisms forfermentative production of these compounds and other diamines or theirimmediate precursors have been reported. Typically, processes for theirfermentation and isolation require acids and bases that generate saltby-products.

During the fermentation process, which utilizes carbon dioxide, at leastone or more of diamine carbonate, diamine bicarbonate, and/or diaminebis-bicarbonate (referred to herein as the diamine “Carbonates”) and,optionally diamine carbamate or diamine biscarbamate (referred to hereinas the diamine “Carbamates”, are produced. The disclosed process furtherprovides increased yields of the diamines and improves the purificationof the desired diamine using organic solvent-based extraction.

FIG. 1 schematically illustrates one embodiment of the invention thatincludes the process steps of culturing a microbial organism, using thecultured microbial organism to produce one or more HMD-Carbonates and/orHMD Carbamates, producing an HMD neutral-charge or free base having theformula H2N—(CH2)6-NH2 and having a higher solubility in hydrophobicorganic solvents compared the HMD salts or HMD Carbonates and/orCarbamates, and isolating the HMD neutral-charge or HMD free base.

FIG. 2 schematically illustrates another embodiment of the inventionthat includes the process steps of providing an impure, biosyntheticsource of HMD, producing one or more HMD-Carbonates and/or Carbamatesthat are charged compounds in the presence of CO2, isolating orseparating the HMD Carbonates and/or Carbamates from undesiredbyproducts or materials, producing an HMD neutral-charge or HMD freebase compound having the formula H2N(CH2)6NH2 and having a highersolubility in hydrophobic organic solvents compared to the charged HMDCarbonates and/or Carbamates, and isolating the HMD neutral-charge orHMD free base.

FIG. 3 schematically illustrates an embodiment of a fermentation systemthat may be used to produce HMD. A genetically engineered microorganismis cultured or grown in reaction vessel 10 in a suitable culture orfermentation medium comprising a nitrogen source and a carbon source. Inone embodiment of the invention, the typical culture or fermentationmedium and growth conditions are set out below in Example 3. During thefermentation of the genetically engineered microorganism to produce thedesired diamine (e.g. HMD), carbon dioxide is used to control the pH ofthe cultured medium. The carbon dioxide used may be producedmetabolically by the microorganism, artificially or added from anexternal source. In an embodiment, the growth condition of themicroorganism and the concentration of CO2 in the culture orfermentation medium are controlled so that the pH is maintained at apredetermined level during selected times during the fermentation cycle.Over the course of the fermentation process, the pH will rise from aboutneutral to a stable pH of 8.5 due to, for example, the buffer created byHMD and carbon dioxide. Upon completion of the fermentation or whenHMD-Carbonates and/or Carbamates are produced, the cells may be removed(e.g. by membrane filtration 20) to separate the crude or impure aqueoussolution containing charged HMD-Carbonate and/or Carbamate materials,such as HMD salts, from undesired by-products that will be contained inthe retentate. After at least cell removal, the filtered, aqueousfermentation solution containing at least charged HMD-Carbonates and/orCarbamates materials can be stripped of carbon dioxide with inert gas orsteam via steam stripping. Steam can be added from an external source,or generated “in situ” by boiling the broth under elevated temperatureand pressure in reactor 30. Removal of CO2 will generateneutrally-charged or free base HMD in aqueous solution. By removing thecarbon dioxide, the pH of the solution is raised to a point wheresolvent extraction can be used to remove HMD in its free base form. Theneutral-charged or free base HMD may be extracted from the aqueoussolution in extractor 40 which separates the organic components from theaqueous raffinate. The organic components containing at least solventand HMD can be separated in distillation column and the distilled HMDcan be further purified by distillation in distillation column 60.Recovered CO2 from reactor 30 and the recovered solvent fromdistillation column may be recycled in the described process to improveprocess and economic properties of the illustrated fermentation process.

Culturing a microorganism in medium under suitable conditions and for asufficient period of time results in the formation of one or morediamine Carbonates and/or Carbamates. The produced compounds in thecultured medium include at least 40% Carbonates and/or Carbamates in thecultured medium. In other embodiments, the Carbonates and/or Carbamatescan be at least 50%, 60%, 70%, 80, 90% or 99.9% in the cultured medium.As defined above, this means that desired diamine (e.g. HMD) carbonatesor carbamates comprise at least 40% or more of all carbonates and/orcarbamates in the cultured medium.

Culture Medium

Depending on the desired microorganism or strain to be used, theappropriate culture medium may be used. For example, descriptions ofvarious culture media may be found in “Manual of Methods for GeneralBacteriology” of the American Society for Bacteriology (Washington D.C.,USA, 1981). As used here, “medium” as it relates to the growth sourcerefers to the starting medium be it in a solid or liquid form. “Culturedmedium”, on the other hand and as used here refers to medium (e.g.liquid medium) containing microbes that have been fermentatively grownand can include other cellular biomass. The medium generally includesone or more carbon sources, nitrogen sources, inorganic salts, vitaminsand/or trace elements.

Exemplary carbon sources include sugar carbons such as sucrose, glucose,galactose, fructose, mannose, isomaltose, xylose, pannose, maltose,arabinose, cellobiose and 3-, 4-, or 5-oligomers thereof. Other carbonsources include alcohol carbon sources such as methanol, ethanol,glycerol, formate and fatty acids. Still other carbon sources includecarbon sources from gas such as synthesis gas, waste gas, methane, CO,CO₂ and any mixture of CO, CO₂ with H₂. Other carbon sources can includerenewal feedstocks and biomass. Exemplary renewal feedstocks includecellulosic biomass, hemicellulosic biomass and lignin feedstocks.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare disclosed, for example, in U.S. Patent Application Publication No2009/0047719, filed Aug. 10, 2007. Any of these conditions can beemployed with the microbial organisms as well as other anaerobicconditions well known in the art.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of theproducts can be obtained under anaerobic or substantially anaerobicculture conditions.

An exemplary growth condition for achieving, hexamethylenediamineincludes anaerobic culture or fermentation conditions. In certainembodiments, the microbial organism can be sustained, cultured orfermented under anaerobic or substantially anaerobic conditions.Briefly, anaerobic conditions refer to an environment devoid of oxygen.Substantially anaerobic conditions include, for example, a culture,batch fermentation or continuous fermentation such that the dissolvedoxygen concentration in the medium remains between 0 and 10% ofsaturation. Substantially anaerobic conditions also includes growing orresting cells in liquid medium or on solid agar inside a sealed chambermaintained with an atmosphere of less than 1% oxygen. The percent ofoxygen can be maintained by, for example, sparging the culture with anN2/CO2 mixture or other suitable non-oxygen gas or gases.

The culture conditions can be scaled up and grown continuously formanufacturing hexamethylenediamine. Exemplary growth procedures include,for example, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. All of these processes are well known in the art.Fermentation procedures are particularly useful for the biosyntheticproduction of commercial quantities of, hexamethylenediamine. Generally,and as with non-continuous culture procedures, the continuous and/ornear-continuous production of hexamethylenediamine will includeculturing a hexamethylenediamine producing organism on sufficientnutrients and medium to sustain and/or nearly sustain growth in anexponential phase. Continuous culture under such conditions can include,for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally,continuous culture can include 1 week, 2, 3, 4 or 5 or more weeks and upto several months. Alternatively, the desired microorganism can becultured for hours, if suitable for a particular application. It is tobe understood that the continuous and/or near-continuous cultureconditions also can include all time intervals in between theseexemplary periods. It is further understood that the time of culturingthe microbial organism is for a sufficient period of time to produce asufficient amount of product for a desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of hexamethylenediamine can be utilizedin, for example, fed-batch fermentation and batch separation; fed-batchfermentation and continuous separation, or continuous fermentation andcontinuous separation. Examples of batch and continuous fermentationprocedures are well known in the art.

The culture medium at the start of fermentation may have a pH of about 5to about 7. The pH may be less than 11, less than 10, less than 9, orless than 8. In other embodiments the pH may be at least 2, at least 3,at least 4, at least 5, at least 6, or at least 7. In other embodiments,the pH of the medium may be about 6 to about 9.5; 6 to about 9, about 6to 8 or about 8 to 9.

CO2 Sources and Species

As noted above, to produce the desired diamine (e.g. HMD) and to controlpH of the culture medium CO2 is added. The source of CO2 can take theform of CO2, carbonate, bicarbonate or carbonic acid, for example. Inone embodiment, CO2 may be externally added to the cultured medium. Inother embodiments, CO2 may be produced by the microorganism such as byrespiration or as a by-product. For example, the respiration CO2 may beformed from the conversion of the tricarboxylic acid (TCA) cycle, viathe glyoxylate shunt, the pentose phosphate pathway (e.g. gnd(6-phosphogluconate dehydrogenase that converts 6-phosphogluconate toribuloase-5-phosphate and CO₂)) or the Entner Duodoroff pathways. Inother embodiments, the by-product CO2 may be formed from acetate,ethanol, succinate, 3-oxoadipate or 3-hydroxyadipate.

Culturing the microorganism under suitable conditions and sufficientperiods of time also results in the microorganism forming one or moreCO2 sources that include 002, carbonate, bicarbonate or carbonic acids.In one embodiment, the microorganism forms CO2 stoichiometrically withthe diamine.

Stoichiometric CO2 as referred here is the amount of CO2 associated withthe formation of the product from a given substrate based on thestoichiometry. One exemplary stoichiometry for the production of HMDfrom glucose is:1.47C6H12O6+0.321O2+2NH3→C6H16N2+2.821CO2+3.821H2OThe stoichiometric CO2 amount produced per mole of HMD is 2.821 moles.Any CO2 produced higher than this amount is either produced byrespiration or byproduct formation.

Some sources of respiratory CO2 are the TCA cycle, glyoxylate shunt,pentose phosphate pathway (e.g. zwf) and the Entner-Duodoroff pathwayamong others. These pathways produce NAD(P)H that can then be used viathe electron transport chain to form ATP. Byproduct CO2 is defined asthe CO2 produced due to the formation of byproducts. For example, 2moles of acetate are produced from every mole of glucose and this isassociated with the release of 2 moles of CO2. Therefore, the byproductCO2 associated with the formation of each mole of acetate is 1 mole.

In another example, the production of HMD from methanol can havemultiple stoichiometries, depending upon whether only the oxidative TCAbranch is used to make HMD, compared if both the oxidative and thereductive TCA branches are used to make HMD6.31CH₃OH+2NH₃+0.97O₂+C₆H₁₆N₂+7.62H₂O+0.31CO₂7CH₃OH+2NH₃+2O₂+C₆H₁₆N₂+9H₂O+CO₂

In the case where CO2 is used to drop the pH (e.g. to 7), the solubilityof the medium towards CO2 may be enhanced by raising the back pressureat the top of the fermentor to at least 2, but not exceeding 10 bar toincrease the solubility of carbon dioxide. In other embodiments, thetemperature may be lowered to enhance solubility of the medium for 002.In one embodiment, the temperature is lowered below 37° C. to increasethe solubility of 002.

In one embodiment, a microorganism producing the diamine is cultured ina liquid medium in a fermenter, wherein an inlet gas including carbondioxide is fed into the fermenter and the back pressure at the top ofthe fermentor is raised to at least 2, but not exceeding 10 bar toincrease the solubility of carbon dioxide.

In another embodiment, the process for the fermentative production of adiamine, wherein a microorganism producing the diamine is cultured in aliquid medium in a fermenter, wherein an inlet gas including carbondioxide is fed into the fermenter and the temperature is lowered below37° C. to increase the solubility of CO2.

In some embodiments, an enzyme carbonic anhydrase (CA) may be added tothe fermentation broth or medium to catalyze or enhance the formation ofdiamine Carbonates and/or diamine Carbamates (e.g. HMDA Carbonates) byincreasing the amount or rate of gaseous CO2 converted to soluble ion,thus a greater amount or availability of soluble ion is available to thediamine or HMD.

Carbonic anhydrase catalyzes a reversible reaction. In the forwardreaction, CA combines carbon dioxide with water:

and H2CO3 dissociates to form bicarbonate (HCO3-) and a proton. In thereverse, reaction, CA combines bicarbonate and a proton to providecarbon dioxide and water. Therefore, the reversible reaction, inparticular the reverse reaction can be used to strip or release the CO2after the DA carbonate is formed. In some embodiments, the CA may beused to hydrate CO2 in the form of bicarbonate and proton, which in turnmay be converted to a DA. Depending on the direction of the reaction,certain suitable conditions can be selected that favor the absorption ofcarbon dioxide into a solution (e.g., via hydration of carbon dioxide tobicarbonate) and/or the desorption of carbon dioxide from a solution(e.g., via dehydration of bicarbonate to carbon dioxide and water).

The carbonic anhydrase may be provided exogenously by directly providingthe CA to the fermentation solution or may be introduced via amicroorganism capable of producing carbonic anhydrase. Carbonicanhydrase may also be provided as a recombinant or engineered CA. Therecombinant CA may be part of a microorganism that includes a diaminepathway (e.g. HMD synthesis pathway) or may be introduced by anothermicroorganism capable of CA expression. The microorganism's native CAmay be used, for example by overexpressing it or engineering it to beexcreted into the fermentation broth or solution or be secreted into themicroorganism's periplasm. The CA may be of the EC 4.2.1.1. enzymeclass. The CA may be that of an Escherichia, for example Can gene orb1026 (KEGG designation) or other host strain, including strains listedherein. In some embodiments the CA may be obtained from the genusMethanobacterium, Desulfovirbio, Methanosarcina, Thiomicrospira,Acetobacterium, Clostridium, Methylobacterium, Rhizobium, Rhodobacter,Rhodospirillum, Staphylococcus, Methanococcus, Methanosaeta,Methanospirillum, Sulfolobus. (Smith et al., 1999 PNAS96(26):15184-15189.

Exemplary organisms from which the CA may be obtained include Neisseriagonorrhoeae (Jo et al., 2013 Appl. Environ. Microbiol.79(21):6697-6705), Methanosarcina, Thiomicrospira, Acetobacteriumwoodii, Clostridium thermoaceticum, Methylobacterium extorquens,Rhizobium meffloti, Rhodobacter capsulatus, Rhodobacter sphaeroides,Rhodospirillum rubrum, Staphylococcus aureus, Methanococcus jannaschii,Methanosaeta concilii, Methanosarcina barkeri, Methanosarcinathermophila, Methanospirillum hungateii, Sulfolobus solfataricus. (Smithet al., 1999 PNAS 96(26):15184-15189. The CA may be obtained fromNeisseria gonorrhoeae (Jo et al., 2013 Appl. Environ. Microbiol.79(21):6697-6705). In some embodiments, a carbonic anhydrase of aNeisseria gonorrhoeae may be engineered for periplasmic expression in E.coli as reported Jo et al., 2013 Appl. Environ. Microbiol.79(21):6697-6705

In some embodiments, the gene encoding a β type CA may be obtained fromthe genus Desulfovirbio such as Desulfovirbio fructosivorans,Desulfovirbio Tom C, Desulfovirbio magneticus, Desulfovirbioalcholivorans. In still other embodiments, the CA may be obtained fromthe genus Desulfomonile such as Desulfomonile tiedjei, Methanobacterumsuch as Methanobacterium thermoautotrophicum, Metanoacina such asMetanoacina thermophilia, Thiomicrospira such as Thiomicrospiracrunogena.

In other embodiments, the CA may be used as shown in Table A below:

TABLE A Sequence %  Identifiers Identity Sequence gi|506426310|ref|100.0 [Desulfovibrio vulgaris  WP_015946029.1|;gi| str. ‘Miyazaki F’218758438|gb| MRLRFLSALFLVVAMVGTALAGS ACL09337. 1|TGPGIGPDEALQRLKEGNARFVA ETPTRQNLSAKRLATSQHGQTPY ATILSCADSRAPVELIFDEGVGDLFVIRVAGNVAATDEVGTAEYGA DHLNVPLLVVMGHTQCGAVTAVV QGAEVHGSIPMLVAPIVPAVTAVEKSNPKHDRAALVPKVIEANVWQ AIDDTMRQSPIIRARVAAGKLKV VGAIYHIDDGKVEWLGEHPMQARLLNYTSGPAKAHR gi|496399538|ref|  55.1 [Desulfovibrio sp. U5L]WP_009108528.1|;gi| MKRFLAATATMAFLLAMCTAVLA 385733492|gb|SSGGPEVSADEALSRLKEGNTRF EIG53690.1| VSQANVAPHQDAARRHETATGGQHPFATVLSCADSRAPVEVLFDQG VGDLFVVRVAGNVAATDEIGTIE YGAEHLGVPLVVVLAHTKCGAVTAVVKNEPVTENIGKLVAPIVPAV KGVKARFAASDVNEIISRSIEAN MWQAVSDIYAKSPMLKKMAADGKIKVVGALYDIDSGEVHWFGEHPS EGNLLDN gi|760112986|ref|  59.9 [DesulfovibrioWP_043794941.1| fructosivorans] MKRAFAAFAAAVFVAATCALALASSAGPGLTSDEALAKLKEGNDRY VAKASVAPRRDAARRHETATGGQ HPFATVLACSDSRVPVEVVFDQGVGDIFVVRVAGNVAATDEIGTME YGAEHLGVPLIVVMGHTKCGAVS AVVKNEPVTENIGKLVAPIVPAVKSVKARFATANTDELIAKSIEAN VWQAISDIYAKSPLIKKMAAAGK VKVVGALYDIDSGEVHWLGEHPNNAILLGK gi|302490589|gb|  59.9 [Desulfovibrio EFL50494.1|fructosivorans JJ] MMKRAFAAFAAAVFVAATCALAL ASSAGPGLTSDEALAKLKEGNDRYVAKASVAPRRDAARRHETATGG QHPFATVLACSDSRVPVEVVFDQ GVGDIFVVRVAGNVAATDEIGTMEYGAEHLGVPLIVVMGHTKCGAV SAVVKNEPVTENIGKLVAPIVPA VKSVKARFATANTDELIAKSIEANVWQAISDIYAKSPLIKKMAAAG KVKVVGALYDIDSGEVHWLGEHP NNAILLGKgi|759946892|ref|  58.4 [Desulfovibrio WP_043631526.1| sp. TomC]MRRNMTAMTVVIWTLCMATTALA FSGGAGITADEALSRLKEGNTRF VAGAAVTPRQDAARRHETTVGGQHPFATVLACADSRVPVEAIVDQG VGDVFVVRVAGNVANTDEIGTIE YGAEHLGVPLVVVLGHTKCGAVTAVVKGEHVTENIGKLVAPIVPAV AGVKNRFASADLDELINRSIEAN VWQSISDMYANSPLLKKMAADGKLKVVGALYDIDSGDIHWLGEHPS NAKLLGN gi|732991830|gb|  58.3 [DesulfovibrioKHK04204.1| sp. TomC] MTVVIWILCMATTALAFSGGAGI TADEALSRLKEGNTRFVAGAAVTPRQDAARRHETTVGGQHPFATVL ACADSRVPVEAIVDQGVGDVFVV RVAGNVANTDEIGTIEYGAEHLGVPLVVVLGHTKCGAVTAVVKGEH VTENIGKLVAPIVPAVAGVKNRF ASADLDELINRSIEANVWQSISDMYANSPLLKKMAADGKLKVVGAL YDIDSGDIHWLGEHPSNAKLLGN gi|493978453|ref|  55.7[Desulfovibrio WP_006921438.1|;gi| magneticus] 409981683|gb|MKRFVTAFAGAVITISMAGAAMA EKO38218.1| FSGGAGISADEALARLKEGNTRYVAGAAVTPRQDAARRHETATGGQ HPFVSVLSCADSRVPVETVFDQG IGDVFVIRVAGNVANTDEIGTIEYGAEHLGTPLVLVMAHTKCGAVT AVVKGEHVTENIGKLVAPIVPAV ASVKSRFATDDVNELINRSIEANMWQAIADMYAKSPLLKKMAADGK IKVVGALYDIDSGEVHWFGEHPS NANLLGKgi|496471458|ref|  54.7 [Desulfovibrio WP_009180303.1|;gi| sp. FW1012B]357581548|gb| MKRFLAATATMAFLLAMCTAVLA EHJ46881.1|SSGGSEVSADEALSRLKEGNTRF VSQANVAPHQDAARRHETATGGQ HPFATVLSCADSRAPVEVLFDQGVGDLFVVRVAGNVAATDEIGTIE YGAEHLGVPLVVVLAHTKCGAVT AVVKNEPVTENIGKLVAPIVPAVKGIKARFAASDVNEIISRSIEAN MWQAISDIYAKSPMLKKMAADGK IKVVGALYDIDSGEVRWFGEHPSEGSLLDN gi|752616536|ref|  53.8 [Desulfomonile tiedjei] WP_041285901.1|MEAFMKKIAVLFSVICMLGSVFS WAADPAATVSPEEAVKLLKEGNG RFIAGTSQHPNNDLQRRNTTAAQGQHPFVTVLSCSDSRVPVEVLFD RGVGDIFVIRVAGNVANGDEVGS IEYAVDHLGTPLLVILGHTKCGAVTAVVQSAELLGNIIPIGKSIFP AVVAAKKSNPKASGDALINDAIK ANVWQAIEDIYRTSPITAARVKSGKLKVVGALYDIESGNVSWLGSH PKEGGLLSDKGH gi|390622030|gb|  53.8[Desulfomonile tiedjei AFM23237.1| DSM 6799] MKKIAVLFSVICMLGSVFSWAADPAATVSPEEAVKLLKEGNGRFIA GTSQHPNNDLQRRNTTAAQGQHP FVTVLSCSDSRVPVEVLFDRGVGDIFVIRVAGNVANGDEVGSIEYA VDHLGTPLLVILGHTKCGAVTAV VQSAELLGNIIPIGKSIFPAVVAAKKSNPKASGDALINDAIKANVW QAIEDIYRTSPITAARVKSGKLK VVGALYDIESGNVSWLGSHPKEGGLLSDKGH gi|657653962|ref|  58.8 [Desulfovibrio WP_029458402.1|alcoholivorans] MKRLFTATTMLALLLACCALALA SSGGPGLTADEALAKLKEGNMRYVAQASVAPHQDAARRHETATDGQ HPFATILSCADSRVPLEIIFDQG VGDIFAVRVAGNVAAVDEIGTMEYGAEHLGVPLIVVLGHTKCGAVT AVVKNEPVTENIGQLVAPIVPAV KSVKSRFASASLDELINKSIEANVWQAVSDIYAKSPLLKKMAAAGK VKVVGALYDIDSGKVQWFGEHPS NASLLGKgi|506341077|ref|  54.9 [Desulfovibrio WP_015860796.1|;gi| magneticus]239796622|dbj| MKRFVAAFAGAVITFSMAGAAMA BAH75611.1FSGGAGISADEALARLKEGNTRY VAGAAVTPRQDAARRHETATGGQ HPFVSVLSCADSRVPVETVFDQGIGDVFVIRVAGNVANTDEIGTIE YGTEHLGTPLVVVLAHTKCGAVT AVVKGEHVTENIGKLVAPIVPAVASVKSRFASGDLNELINRSIEAN MWQAIADMYAKSPLLKKMAADGK IKVVGALYDIDSGDVHWFGEHPSNANLIGK Escherichia coli  Escherichia coli str. str. K-12 substr,K-12 substr, MG1655]yadF (MG1655]MKDIDTLISNNALWS KMLVEEDPGFFEKLAQAQKPRFLWIGCSDSRVPAERLTGLEPGELF VHRNVANLVIHTDLNCLSVVQYA VDVLEVEHIIICGHYGCGGVQAAVENPELGLINNWLLHIRDIWFKH SSLLGEMPQERRLDTLCELNVME QVYNLGHSTIMQSAWKRGQKVTIHGWAYGIHDGLLRDLDVTATNRE TLEQRYRHGISNLKLKHANHK >b0339;  Escherichia coliNP_414873.1; MKEIIDGFLK FQREAFPKRE GI:16128324] CynTALFKQLATQQ SPRTLFISCS DSRLVPELVT QREPGDLFVI RNAGNIVPSY GPEPGGVSASVEYAVAALRV SDIVICGHSN CGAMTAIASC QCMDHMPAVS HWLRYADSAR VVNEARPHSDLPSKAAAMVR ENVIAQLANL QTHPSVRLAL EEGRIALHGW VYDIESGSIA AFDGATRQFVPLAANPRVCA IPLRQPTAA GI:157878699 Neisseria gonorrhoeaeHTHWGYTGHD SPESWGNLSE EFRLCSTGKN QSPVNITETV SGKLPAIKVN YKPSMVDVENNGHTIQVNYP EGGNTLTVNG RTYTLKQFHF HVPSENQIKG RTFPMEAHFV HLDENKQPLVLAVLYEAGKT NGRLSSIWNV MPMTAGKVKL NQPFDASTLL PKRLKYYRFA GSLTTPPCTEGVSWLVLKTY DHIDQAQAEK FTRAVGSENN RPVQPLNARV VIE

The CA may be encoded by Desulfovibrio vulgaris. Desulfovibrio vulgariswhich has unique properties of having high activity in 4.2 MN-methyldiethanolamine (MDEA) at elevated temperatures and pH >10. TheD. vulgaris CA has been evolved to be active at 100° C. for long periodsof time (8 weeks) in high concentrations of MDEA for use in carboncapture technology (Alvizo, et. al. 2014 PNAS 111(46): 16436-16441). Insome embodiments, the engineered CA is from Desulfovibrio vulgaris(GenBank accession ACL09337.1 GI:218758438). In some embodiments, theDesulfovibrio vulgaris str “Miyazaki F” carbonic anhydrase has aminoacid substitution to stabilize the carbonic anhydrase activity atelevated temperatures and alkaline pH by including one or moresubstitutions identified as: A56S, T3OR, A40L, A84Q, G120R, T139M, K37R,E68AQ, A95V, Q119M, N145WFC, N213E, A219T, R31P, Q43M, V70I, H124T,H148T, V157A, M170F, H44L, M129F, S144R, Y49F, S126N, D196S, P136R,P174E, D195A, G89A, D96E, V100T, A121Q, A181K, M207A, S216D.

In some embodiments, a CA is encoded by E. coli (EG10176 (EcoCyc), orEG12319 (EcoCyc), Can gene, b1026 (KEGG designation)).

The disclosed enzymes may also be in the form of fusion proteins inwhich the recombinant or engineered CA are fused to antibody tags (e.g.myc epitope), purification sequences (e.g. His tags for binding tometals and cell localization signals (e.g. secretion or excretionsignal). To aid in the expression of the desired protein intoperiplasmic space, a secretion signal may be use such as a Sec tag orTat tag. In preferred embodiments the CA is excreted into the mediarather than secreted to the periplasmic space. In some embodiments, thesecretion or excretion signal is fused to the N-terminus of the proteinby genetically encoding the secretion tag as a fusion to the carbonicanhydrase DNA sequence to aid expression into the periplasmic space orextracellularly excreted from the microorganism (e.g. E coli). Inanother embodiment, CA may be fused to an E. coli protein OmpF that istransported (excreted) to the culture medium (Nagahari et al., 1985 TheEMBO J. 4(13A):3589-3592; Jeong and Lee, 2002 Appl. Environ. Microbiol.68:4979-4985). In still another embodiment CA may be fused to the E.coli protein YebF which as been shown to support protein export to theculture medium, which has an unknown function, but is an extracellularprotein (Zhang et al., 2006 Nat. Biotech. 24:100-10). An N-terminalsecretion signal peptide tag is identified using SignalP 4.1 Server(http://www.cbs.dtu.dk/services/SignalP/).

The carbonic anhydrase may be provided by a genetically engineeredmicroorganism in the fermentation broth. In other embodiments, thecarbonic anhydrase is provided by a genetically engineered microorganismthat produces the DA, optionally excreted to the broth, and optionallypresent in the microorgansim's periplasm. In other embodiments, thecarbonic anhydrase is a native gene or enzyme, optionally engineered forsecretion to the broth, and optionally engineered for secretion to themicroorganism's periplasmic space.

In some embodiments, the CA may be excreted into the fermentation broth,and in other embodiments, the CA may be present in the microorgansim'speriplasm.

Depending on the direction of the reaction or the speed of the reaction,in some embodiments the native CA gene (e.g. E. coli coded CA) sequencemay be overexpressed, for example by modifying its promoter, and orengineered with a secretion or excretion peptide or fusion.

In some embodiments, variants of the CA that are capable of carrying outthe forward and/or reverse reactions are contemplated. The variant canbe a homolog, ortholog, paralogs or genetically engineered, for exampleincreased alkaline pH and heat stability.

CA having an improved property (e.g., thermal stability, solventstability, and/or base stability) that allows them to enhance theforward and/or reverse reaction may be selected for and used. In someembodiments, CA variants that are active and/or stable at highconcentration of DA (e.g. HMD) may be selected for and used. In otherembodiments, CA and variants thereof that are active and/or stable athigh concentrations of C2 to C7 methylene segments such ashexamethylenediamine (HMD), cadaverine, putrescine, ethylenediamine andheptamethylenediamine may selected and used.

Depending on the conditions selected, the CA may be a thermostable CA ormay be a alkaline pH stable CA, or both. Preferably the alkaline pH isabout pH 8-13. In some embodiments, the pH may range from pH 8-13, pH8.5 to 13, pH 9-13, pH 10-13, pH 8-12, pH 8.5-12, pH 9-12, pH 8-11, pH8.5-11, pH 9-11, pH 10-11 and pH 10-12. Temperature and basic pHstability can be useful if the CA is used in the step or steps forrelease of CO2 from the DA Carbonate or Carbamate. This step can resultin increase in pH as CO2 is released and free base formed. Additionallyin some embodiments temperatures above room temperature and abovetypical fermentation temperatures may be used to facilitate release ofCO2.

Depending on whether the forward reaction or the reverse reaction isfavored, the fermentation broth may be provided with CA enzymes thathave different activity. In some embodiments, the CA may have optimalactivity for the forward reaction and in other embodiments the CA enzymemay have optimal activity for the reverse reaction. In some embodiments,a mixture of different enzymes having varying optimal activities and/orimproved properties as disclosed herein may be used.

In some embodiments, the method can be carried out wherein the carbonicanhydrase has the improved property at least 1.2-fold, at least1.3-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 25-fold increasedactivity of hydrating carbon dioxide or dehydrating bicarbonate undersuitable conditions. Accordingly, in some embodiments, the suitableconditions used in the method can comprise a concentration of thecarbonic anhydrase polypeptide of from about 0.1 g/L to about 10 g/L,about 0.25 g/L to about 7.5 g/L, about 0.5 g/L to about 5 g/L, less than10 g/L, less than about 5 g/L, or less than about 2.5 g/L.

In some embodiments, the CA may be provided exogenously. In otherembodiments, the CA enzyme may be provided directly into the broth orfermentation solution. In some embodiments, the CA is provided by agenetically engineered microorganism in the fermentation broth orsolution, where the CA can be excreted into the broth or may be presentin the microorganism's periplasm. In other embodiments, the enzyme maybe provided immobilized on particles. Recombinant carbonic anhydrasepolypeptide may be immobilized on a surface, for example wherein theenzyme is linked to the surface of a solid-phase particle in thesolution. Methods for linking (covalently or non-covalently) enzymes tosolid-phase particles (e.g., porous or non-porous beads, or solidsupports) such that they retain activity for use in bioreactors arewell-known in the art. Methods for treating a gas stream usingimmobilized enzymes are described in e.g., U.S. Pat. No. 6,143,556, U.S.patent publication no. 2007/0004023 A1, and PCT publicationsWO98/55210A1, WO2004/056455A1, and WO2004/028667A1, each of which ishereby incorporated by reference herein.

Accordingly, in some embodiments, the methods for enhancing the releaseof CO2 from a solution of DA Carbonates or DA Carbamates can be carriedout where the engineered carbonic anhydrase polypeptide is immobilizedon a surface, for example where the enzyme is linked to the surface of asolid-phase particle (e.g., beads). In some embodiments, the methodsusing immobilized polypeptides can be carried out where the methodfurther includes a step of isolating or separating the immobilizedcarbonic anhydrase from the broth or fermentation solution. Afterseparating the immobilized carbonic anhydrase from the broth orfermentation solution, the broth or fermentation solution can be treatedto conditions that may inactivate the enzyme, e.g., desorption of CO2 athigh temperatures. Further, the separately retained immobilized enzymecan be added to another solution and reused. CO2:DA Ratios

Under the disclosed process, the microorganism (e.g. geneticallyengineered microorganism) forms CO2 and DA (e.g. HMD) in the ratio ofabout 0.05 to 1 to about 5 to 1 to about 7:1. Other suitable ratiosinclude 0.2 to 1 to about 3 to 1. In other embodiments, the ratio of CO2to DA (e.g. HMD) include about 0.05 to 1 to about 3 to 1; about 0.05 to1 to about 2.5 to 1; about 0.05 to 1 to about 2 to 1; about 0.05 to 1 toabout 1.5 to 1; about 0.05 to 1 to about 1 to 1.

The disclosed ratios may be determined by measuring the CO2 in the formof the total dissolved inorganic carbon (DIC) in the cultured medium.The DIC, which include the Carbonates and/or Carbamates may be measured,for example by the “Handbook of Methods for the Analysis of the VariousParameters of the Carbon Dioxide System in Sea Water.” Prepared for theU.S. Department of Energy, Special Research Grant Program 89-7A: Globalsurvey of carbon dioxide in the oceans. Version 2—September 1994 Editedby Andrew G. Dickson & Catherine Goyet (referred to as the Handbook).For example, the DIC may be measured by the “SOP 2: Determination oftotal dissolved inorganic carbon in sea water, p. 1-18” on pages 38-55of the Handbook.

In other embodiments, the fraction of DIC, which is DIC over TotalDissolved Counter Anions (TDCA) may be measured. The TDCA is the sum ofDIC and other anions. The other anions (e.g., Cl⁻, SO⁻², PO₄ ⁻³, NO₃ ⁻,NO₂ ⁻) other than the DIC can be determined using any suitable methodsuch as ion exchange chromatography. For example, a commerciallyavailable ion exchange chromatography by DIONEX with conductivitydetector (and ion suppressor) may be used.

The produced compounds in the cultured medium include have a DIC/TDCAvalue of at least 40%. In other embodiments, the DIC percentage can beat least 50%, 60%, 70%, 80, 90% or 99.9% in the cultured medium. In someembodiments, the DIC is at least 40%, 50%, 60%, 70%, 80, 90% or 99.9% ofTDCA in the cultured medium at pH 9.

Fermentation pH

As noted above, the starting culture medium may have a pH of about 5 toabout 7. As the microorganism grows on the culture medium and producesthe desired diamine (e.g. HMD-Carbonates and/or HMD Carbamates) andbefore the diamine Carbonates and/or Carbamates are converted to thediamine free base, the pH of the cultured medium may be less than 11,less than 10, less than 9, or less than 8. In other embodiments the pHmay be at least 2, at least 3, at least 4, at least 5, at least 6, or atleast 7. In other embodiments, the pH of the medium may be about 6 toabout 9.5; about 6 to about 9, about 6 to about 8, about 6.5 to about7.5, about 7.5 to about 9.5 or about 8 to about 9.

While the medium (i.e. the starting medium) may be adjusted withinorganic acids, bases or buffers to adjust pH, the cultured medium issubstantially free of buffer, substantially free of inorganic or organicacid, or externally added inorganic or organic acid. As used here,“substantially free” relates to the cultured medium. In other words,salts, buffers, acids (not including CO2) or bases may be used tocontrol pH of the starting medium. If such salts, buffers, acids (notincluding CO2) or bases are used to adjust pH fluctuations asfermentation and growth of the organism progresses, minimal amounts areused. But the salts, buffers, acids (not including CO2) or bases are notused to neutralize the diamine Carbonate and/or Carbamate. It should beunderstood the microorganism will produce by-products such as acetates,succinates, other salts and/or organic acids. Adjusting pH duringfermentation is by the use of carbon dioxide that may be addedexternally or generated by the microorganism's growth.

Release of CO2 & Diamine Free Base

Once the diamine-Carbonates and/or Carbamates are formed, the diamine isobtained by converting to diamine free base, e.g. HMD free base. In someembodiments, the DA-Carbonates and/or Carbamates are first separatedfrom the microorganism in the cultured medium before converting to DAfree base. Examples of converting DA-Carbonates and/or Carbamates to DAfree base include by heat, vacuum, ion exchange or electrodialysis. Inone embodiment, the diamine is an HMD free base that may be converted byreleasing carbon dioxide. In some embodiments, a carbonic anhydraseenzyme (as described more fully above in the context of the forwardreaction) may be provided to enhance the release of carbon dioxide froma solution of DA Carbonates or DA Carbamates by converting a bicarbonateand/or carbonate ions to carbon dioxide. In such embodiments, theenzymes may be provided exogenously, for example, as an engineeredenzyme that may be part of a microorganism or exogenously added. In someembodiments, the engineered CA may be engineered to be excreted into thefermentation broth or fermentation solution. In other embodiments, theengineered CA may be engineered to be in the microorganism's periplasm.In some embodiments, the DA-Carbonates and/or Carbamates are firstseparated from the microorganism in the cultured medium beforeconverting to DA free base. In such embodiments, the CA may be providedby exogenously adding the CA. In other embodiments, the CA may beimmobilized. In other embodiments, the CA is part of an engineeredmicroorganism capable of providing carbonic anhydrase activity. In otherembodiments, the carbonic anhydrase activity is provided by anengineered microorganism that includes a DA synthesis pathway such as aHMD synthesis pathway and the carbonic anhydrase activity.

If heat is used to convert diamine Carbonates and/or Carbamates (e.g.HMD) to diamine free base, the temperatures include greater than 70° C.,greater than 80° C. or greater than 105° C. In some embodiments, thetemperature may be greater than 200° C. In still other embodiments, thetemperature may be about 315° C. In some embodiments, the temperature isless than 315° C., less than 250° C. or less than 215° C. In still otherembodiments, the temperature may be greater than 20° C., greater than30° C., or greater than 40° C. and where a vacuum is used. In someembodiments, the diamine Carbonates and/or Carbamates converted to freebase at temperatures disclosed above may be HMD. CA may also be added tothe heating step to enhance the release of carbon dioxide from asolution of DA Carbonates or DA Carbamates by converting a bicarbonateand/or carbonate ions to carbon dioxide and free base. Accordingly, insome embodiments, CA having an improved property (e.g., thermalstability, solvent stability, improved stability or activity in highconcentrations of DA and/or base stability) that favors the reversereaction is provided. Thus, in some embodiments the method of carbonicanhydrase catalyzed reverse reaction may be carried out at a temperaturegreater than 70° C., greater than 80° C. or greater than 105° C. In someembodiments, the temperature may be greater than 200° C. In still otherembodiments, the temperature may be about 315° C. In some embodiments,the temperature is less than 315° C., less than 250° C. or less than215° C. In still other embodiments, the temperature may be greater than20° C., greater than 30° C., or greater than 40° C. and where a vacuumis used, greater than 70° C., greater than 80° C. or greater than 105°C. In some embodiments, the temperature may be greater than 200° C. Instill other embodiments, the temperature may be about 315° C. In someembodiments, the temperature is less than 315° C., less than 250° C. orless than 215° C. In still other embodiments, the temperature may begreater than 20° C., greater than 30° C., or greater than 40° C. andwhere a vacuum is used.

Conversion may also be carried out in a vacuum such as lower thanatmospheric pressure (e.g. 0.01 to 1 atm). In other embodiments, thepressure may include pressure of about 1 to about 10 bar (within thevessel, not the inlet air pressure) or about 1 to about 3 bar. Whentemperature and pressure are used in combination, the pressure may befrom about 1 to about 3 bar. In some embodiments, the temperature isless than 315, less than 250 or less than 215.

The released CO2 may be recycled back into the system (e.g. into thecultured medium).

Other examples of converting diamine-Carbonates and/or Carbamates (e.g.HMD) to diamine free base (e.g. HMD free base) include sparging with gas(e.g. air, or inert gas such as nitrogen or helium) or steam stripping.The steam can be added from an external source, or generated in situ byboiling the broth. In some embodiments converting diamine (e.g. HMD)Carbonates and/or Carbamates to free base include by heat and spargingwith gas. In one embodiment, stripping may be carried out at a pressureof about 1 to about 10 bar. In some embodiments, the converting step canresult in about at least 20% to about at least 99% diamine free base. Inother embodiments, the converting step can result in about at leastabout 20-30%, 30-40%, or 40-50%.

When sufficient heat is added to generate steam from the medium in situin a stripping column, both water and carbon dioxide can be removed insufficient amounts to obtain a solution concentrated in diamine (e.g.HMD) free base that allows efficient subsequent diamine recovery. Insome embodiments, CA is also added to enhance the release of carbondioxide from a solution of DA Carbonates or DA Carbamates by convertinga bicarbonate and/or carbonate ions to carbon dioxide. Increasing theefficiency of CO2 removal from the DA Carbonate salt solution (e.g.HMD-bicarbonate salt solution) can lower purification costs by reducingthe size of the stripping column.

In some embodiments, the diamine recovered as diamine free base can begreater than 40% or greater than 50%. In still other embodiments, thediamine recovered can be greater than 50% in free base form from thestripping step under ambient pressure, air sparge, and high temperature(e.g. less than 315° C., less than 215° C., or around 115° C.).

The addition of strong base (e.g. sodium or calcium hydroxide) may beadded to raise the pH after diamine (e.g. HMD) free base is generatedand CO2 is removed, thereby improving the extraction. If calciumhydroxide is used, a carbonate precipitate will form, which can then beseparated from the liquid phase.

Solids Removal Prior to Conversion

Before converting the diamine (e.g. HMO) Carbonates and/or Carbamates tothe diamine free base, solids can be separated from the cultured medium.Such solids may include cells and other biomass by products andimpurities from the cultured medium. The resultant liquid fraction maybe enriched in the diamine (e.g. HMD) Carbonates and/or Carbamates.

Separation may be achieved by centrifugation, filtration, rotary drum orcombinations thereof. Exemplary centrifugation may be by a disc-stackcentrifuge or decanter or solid bowl centrifuge. It should be understoodthat any combination of centrifugation types or configurations andnumber of centrifugations may be used to achieve the desired solidsseparation from the culture medium. If solids are not separable bycentrifugation or additional separation is required, separation byfiltration may be used. Filtration may be achieved by ultrafiltration.

Water Reduction or Removal

In some embodiments, water may be removed or reduced after the solidsremoval and prior to conversion. Any known suitable process for waterremoval or reduction may be used such as for example, evaporation,reverse osmosis, or electrodialysis.

In other embodiments, water may be removed prior to the step ofisolating the diamine free base. One benefit of removing water beforethe isolation step can be an increase in pH. Methods to reduce or removewater as disclosed above in connection with water reduction or removalafter solids removal and prior to conversion can also be used. Theamount of water removal (e.g. upper limit of water removal) can dependon the solubility limit of a medium component or byproduct or thediamine salt or carbamate. In one embodiment, the water removal isdependent on whether it prevents insolubility of a medium component orbyproduct, including the diamine salt or carbamate

Evaporation may be carried out with multiple effects evaporator, thermalvapor recompression or mechanical vapor recompression. An evaporator isa heat exchanger in which a liquid is boiled to give a vapor that isalso a low pressure steam generator. This steam can be used for furtherheating in another evaporator called another “effect.” Thus, forexample, two evaporators can be connected so that the vapor line fromone is connected to the steam chest of the other providing a two, ordouble-effect evaporator. This configuration can be propagated to athird evaporator to create a triple-effect evaporator, for example.

In one embodiment, the amount of water removed is 10 wt percent. Theremoved water can be further recovered and recycled such as in theculturing process of step a) or as shown in FIG. 4.

Simultaneous Removal of Water and Carbon Dioxide

Water removal or reduction allows for a smaller solvent extractioncolumn and less solvent, and the removal of carbon dioxide enablesalkalization of the diamine composition (e.g. filtration permeate) toincrease solvent extraction efficiency. In one embodiment describedabove, an evaporator is employed to remove sufficient water (and alsoenables removal of CO2) followed by a stripping column to remove anyresidual carbon dioxide (and can also remove water). Simultaneousremoval of water and carbon dioxide in a single step to a pointsufficient for subsequent extraction has the benefit of reducing costsand downtime associated with multiple steps. Accordingly, in anotherembodiment described above, a single step or unit operation is used toremove sufficient water and carbon dioxide (e.g. DIC) to enhancedownstream diamine recovery, such as by solvent extraction. Accordingly,a step or unit operation (e.g. water evaporator or stripping column) andits associated costs of equipment, maintenance, use and risk of downtimemay be absent or reduced. In one embodiment, the simultaneous water andcarbon dioxide removal enhances the downstream recovery of HMD.

Water removal will also enable carbon dioxide stripping. Conditions forwater removal can allow sufficient carbon dioxide removal obviating aneed for a separate CO2 removal step. Thus in one embodiment thesimultaneous removal of water and CO2 is effectively achieved by eithera stripping unit or an evaporator unit. For example, an evaporator, e.g.a multi-effect evaporator, a mechanical vapor recompressor, can be usedto remove sufficient water and carbon dioxide to obtain a solutionconcentrated in diamine free base that allows efficient subsequentdiamine recovery. For example, in FIG. 4, step 4 or 5 can be absent whenthe retained step achieves sufficient removal of both water and carbondioxide. As demonstrated in the Examples, use of an evaporator step canbe advantageous compared to a stream stripping step.

It should be understood that the carbonic anhydrase may be present in astep or steps for releasing carbon dioxide and generating free DA baseto enhance the release of carbon dioxide from a solution of DACarbonates or DA Carbamates. In some embodiments, the CA may be presentin the water removal or evaporation step, in other embodiments, the CAmay be present in the CO2 stripping step, and in still otherembodiments, the CA may be present in both the water removal orevaporation step and the CO2 stripping step.

Isolation of Diamine Free Base

Once converted, the DA (e.g. HMD) free base may be isolated from thecultured medium by extraction with an organic solvent. The isolated DAis separated from the organic solvent by a process such as distillation.Exemplary extraction solvents include alcohols, amines, ethers, alkanesand ketones. Exemplary extraction alcohols include C4 to C8 monohydricalcohols. In some embodiments, the extraction alcohols include hexanol,particularly 1-hexanol, isopentanol, or cyclohexanol, toluene or ethylether or mixtures thereof. Alkanes are suitable solvents as demonstratedin the Examples, particularly when HMD is the diamine. Alkanes,specifically hexane, may be used because of their extremely low watersolubility. Hexane extracted little if any water and provided reasonablerecovery of the available free base. Alkanes are therefore suitablesolvents for use in recovery of diamine free base (e.g. HMD). Suitablealkanes include C5-C12, linear or branched. In one embodiment, both thediamine to be extracted and the alkane selected as solvent may have thesame number of carbon atoms. Heptane is another suitable alkane,especially for HMD, which is further supported by the in silico modelingstudy below. Isomers of hexane and heptane are suitable. Hexane isomersare 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, and2,3-dimethylbutane. Heptane isomers are 2-methylhexane, 3-methylhexane,2,2-dimethylpentane, 2,3-dimethylpentane, 2,4-dimethylpentane,3,3-dimethylpentane, 3-ethylpentane and 2,2,3-trimethylbutane. In someembodiments, the DA (e.g. HMD) free based can be directly distilled fromthe cultured medium.

Any suitable solvent may be used. In some embodiments, the solvent canhave boiling points higher than HMD free base or desired diamine freebase, lower than water or any boiling point in between HMD free base (ordesired diamine free base) and water (an intermediate boiling point).

The DA (e.g. HMD) free base can be isolated from the medium or aDA-enriched fraction (e.g. when solids and/or water is removed beforeisolating) using an extraction solvent to provide an aqueous phase and aDA-free-base-containing organic phase.

The organic phase (extract) can include at least 10%, at least 20%, atleast 30%, at least 40%, or at least 50% by weight DA (e.g. HMD) in freebase, Carbonate, and/or Carbamate form, but predominantly in free baseform. Depending on the number of extractions, in some embodiments, theDA (e.g. HMD) in the extract can be greater than 90% by weight.

The amount of DA free base that is extracted is about greater than 90%by weight. In some embodiments, the DA free base is HMD free base thatis greater than 90% by weight.

The efficiency of solvent extraction of diamine, e.g. HMD, free baseincreases with decrease in carbon dioxide concentration such as DIC asshown in the Examples. A decrease in carbon dioxide results in higher pHand higher concentration of recoverable free base form. In someembodiments, the aqueous diamine solution prior to solvent extractioncontains no detectable carbon dioxide, less than 0.01% carbon dioxide,less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or 1% or less than 5% carbondioxide. In some embodiments, the aqueous diamine solution prior tosolvent extraction contains no detectable DIC, less than 0.01% DIC, lessthan 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or 1% or less than 5% DIC. In otherembodiments, the aqueous DA solution prior to solvent extractioncontains no detectable DIC, less than 0.01% DIC, less than 0.1%, 0.2%,0.3%, 0.4%, 0.5% or 1% or less than 5% DIC.

In one embodiment, CO2 produced stoichiometrically with DA, e.g. HMD, byan enzymatic pathway is used to neutralize DA, e.g. HMD, to maintain pHsuitable for fermentation, generally a pH about 9 or lower. If desiredor needed, further pH control can be achieved by supplementing with CO2produced by the microbe as a by-product (e.g. shunt pyruvate to formateto CO2) or with an external source of CO2. The external CO2 can bepurchased or can be CO2 recycled from the fermentation/isolationprocess. Due to the presence of CO2 during fermentation HMD carbonate,HMD bis-bicarbonate, HMD bicarbonate and a small amount of HMD carbamateand HMD biscarbamate are formed. At the end of fermentation, cells areoptionally removed, and the culture medium is treated under pressure todegrade the HMD carbonate or carbamate compounds (or DA carbonate orcarbamate compounds) releasing, for example, gaseous CO2 and creatingfree base HMD (neutral form 2HN—(CH2)6-NH2) (or DA free base) thatincreases the pH of the cultured medium. The free base or neutral HMD(or DA) may be isolated via solvent extraction. During the process, thereleased CO2 may be recycled. In one embodiment, the disclosed processdoes not require the use of acids to control pH and subsequent additionof base to neutralize the acids to generate solvent extractable freebase HMD (or DA).

In another embodiment of the process, the culture or cultured medium isthermally treated by heating to reflux temperature, for example eitherbatch wise or continually, for example to 90-110° C. at atmosphericpressure, or to a higher temperature at overpressure.

In another embodiment of the process, DA (e.g. HMD) is extracted with anorganic solvent having a miscibility gap with water and stable atalkaline pH, such as in particular a polar, more specifically dipolarprotic, organic solvent. Suitable solvents are as disclosed above.

In one embodiment, DA (e.g. HMD) extraction is and/or subsequent phaseseparation is carried out batchwise at elevated temperature.

The cultured medium, before or after removing the microbial organismsmay be thickened or concentrated by known methods, such as, for example,with the aid of a rotary evaporator, thin-film evaporator, falling filmevaporator, by reverse osmosis or by nanofiltration. If necessary, saltswhich may have precipitated due to the concentration procedure may beremoved, for example by filtration or centrifugation. This concentratedcultured medium can then be worked up in the manner as disclosed hereinto obtain DA (e.g. HMD). For the work up in accordance with thedisclosed process, such a concentration procedure is feasible, but notabsolutely necessary.

According to an embodiment, DA (e.g. HMD) is extracted from the culturedmedium with the aid of an organic solvent. The organic solvent may have,for example, a miscibility gap with water and stable at alkaline pH,such as in particular a polar, dipolar protic, organic solvent. Suitablesolvents are in particular cyclic or open-chain, optionally branchedalkanols having from 3 to 8 carbon atoms, in particular n- andiso-propanol, n-, sec- and iso-butanol, or cyclohexanol, and alson-pentanol, n-hexanol-n-heptanol, n-octanol, 2-octanol and the mono- orpolybranched isomeric forms thereof.

In one embodiment, the extraction and/or subsequent phase separation arecarried out batchwise at an elevated temperature which is limited by theboiling points of water and of the extractant or of possibly formingazeotropes. Using for example, the extractant n-butanol extraction andphase separation could be carried out, for example, at about 25-90° C.or, preferably, at 40-70° C. For extraction, the two phases are stirreduntil the partition equilibrium has been established, for example over aperiod of from 10 seconds to 2 hours, or 5 to 15 min. The phases arethen left to settle until they have separated completely; this takes forexample, from 10 seconds to 5 hours, for example 15 to 120 or 30 to 90minutes, in particular also at a temperature in the range from about25-90° C. or 40-70° C. in the case of n-butanol.

In further embodiments, DA (e.g. HMD) is extracted from the culturedmedium continuously in a multi-stage process (for example inmixer-settler combinations) or continuously in an extraction column.

One of skill in the art may establish the configuration of theextraction columns which can be employed according to the disclosedprocess for the phases to be separated in each case as part ofoptimization routines. Suitable extraction columns are in principlethose without power input or those with power input, for example pulsedcolumns or columns with rotating internals. The skilled worker may also,as part of routine work, select in a suitable manner types and materialsof internals, such as sieve trays, and column trays, to optimize phaseseparation. The basic theories of liquid-liquid extraction of smallmolecules are well known (cf. e.g. H.-J. Rehm and G. Reed, Eds., (1993),Biotechology, Volume 3 Bioprocessing, Chapter 21, VCH, Weinheim). Theconfiguration of industrially applicable extraction columns isdescribed, for example, in Lo et al., Eds., (1983) Handbook of SolventExtraction, John Wiley & Sons, New York. Explicit reference is made tothe disclosure of the textbooks above.

After phase separation, DA (e.g. HMD) is isolated and purified from theDA-containing extract phase in a manner known per se. Possible measuresof recovering DA (e.g. HMD) are in particular, without being limitedthereto, distillation, precipitation as salt with suitable organic orinorganic acids, or combinations of such suitable measures.

Distillation

Distillation may be carried out continuously or batchwise. A singledistillation column or a plurality of distillation columns coupled toone another may be used. Configuring the distillation column apparatusand establishing the operational parameters are the responsibilities ofthe skilled worker. The distillation columns used in each case may bedesigned in a manner known per se (see e.g. Sattler, ThermischeTrennverfahren [Thermal separation methods], 2nd Edition 1995, Weinheim,p. 135ff; Perry's Chemical Engineers Handbook, 7th Edition 1997, NewYork, Section 13). Thus, the distillation columns used may haveseparation-effective internals, such as separation trays, e.g.perforated trays, bubble-cap trays or valve trays, arranged packings,e.g. sheet-metal or fabric packings, or random beds of packings. Thenumber of plates required in the column(s) used and the reflux ratio areessentially governed by the purity requirements and the relative boilingposition of the liquids to be separated, with the skilled worker beingable to ascertain the specific design and operating data by knownmethods.

In some embodiments, the distillation step substantially removes waterand solvent. The temperature of distillation can be below 170 degreesC., below 160 degrees C., below 150 degrees C., or below 140 Degrees C.

Precipitation as salt may be achieved by adding suitable organic orinorganic acids, for example sulfuric acid, hydrochloric acid,phosphoric acid, acetic acid, formic acid, carbonic acid, oxalic acid,etc. In another preferred embodiment, an organic dicarboxylic acid isused, forming a salt which can be used, either directly or afterpurification, for example by recrystallization, in a subsequentpolycondensation to give the polyamide. More specifically, suchdicarboxylic acids are C4-C12-dicarboxylic acids.

The organic DA (e.g. HMD) phase produced in the extraction procedure mayalso be worked up chromatographically. For chromatography, the DA phaseis applied to a suitable resin, for example a strongly or weakly acidicion exchanger (such as Lewatit 1468 S, Dowex Marathon C, Amberlyst 119Wet or others), with the desired product or the contaminants beingpartially or fully retained on the chromatographic resin. Thesechromatographic steps may be repeated, if necessary, using the same orother chromatographic resins. The skilled worker is familiar withselecting the appropriate chromatographic resins and their mosteffective application. The purified product may be concentrated byfiltration or ultrafiltration and stored at an appropriate temperature.

The identity and purity of the compound(s) isolated may be determined byknown technologies. These include high performance liquid chromatography(HPLC), gas chromatography (GC), spectroscopic methods, stainingmethods, thin layer chromatography, NIRS, enzyme assay ormicrobiological assays. These analytical methods are summarized in:Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova etal. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)Bioprocess Engineer. 19:67-70. Ullmann's Encyclopedia of IndustrialChemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp.540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology,John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC inBiochemistry in: Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 17.

The disclosed process may include various combinations of steps orprocesses as depicted in FIG. 4. Referring to FIG. 4, the system andsteps include:

-   -   1. A fermentor or any vessel in which a microorganism may be        cultured or grown in a suitable medium under suitable conditions        and for a sufficient period of time to form one or more of        diamine Carbonates and/or Carbamates in a cultured medium in the        presence of carbon dioxide, carbonate, bicarbonate or carbonic        acid.    -   2. Microbiol Heat Kill/Conversion: Many processes require        inactivation of the microbial culture post-fermentation. Once        the diamine Carbonates and/or Carbamates are formed, they may be        converted to the free base where CO2 is released if        sterilization/heat kill step occurs at elevated temperatures.    -   3: Solids removal: The solids from the cultured medium may be        optionally removed before the diamine Carbonates and/or        Carbamates are converted to release CO2.    -   4: Conversion (all possible ways to release CO2). The released        CO2 may be recycled to fermentor.    -   5: Water Removal: removing water from the DA free base mixture,        and optional recycling water and/or carbon dioxide to the        fermentor;    -   6: Solvent Extraction, extracting the DA mixture with organic        solvent in an extractor to form an organic phase DA solution and        aqueous raffinate, and optionally recycling the aqueous        raffinate to the conversion step;    -   7: Purification: Involves distillation which optionally recycles        organic solvent back to the solvent extraction step, and        purification could involve more distillation columns to purify        the diamine and other steps to remove color forming compounds        and the like.    -   8: Purified DA: the resulting purified DA free base from the        steps above;    -   9: Optional Microbiol Heat Kill, where no CO2 is released    -   10: Optional water removal, recycle of water and possible CO2 if        released removing water from the Carbonates and/or Carbamates        mixture, and optional recycling water and/or carbon dioxide to        the fermentor;    -   11: Optional direct purification from aqueous phase with or        without release of CO2, could involve distillation, ion        exchange, electrodialysis, and other suitable processes or        steps. Possible to recycle water and CO2 if produced in these        steps: optionally directly converting the Carbonates and/or        Carbamates from the cultured medium to form a DA free base        mixture, and optionally releasing of water and/or carbon dioxide        that may be recycled to the fermentor;    -   12: Alkalization (NaOH or CaOH) or other steps to remove        Carbonates from HMD (Ion exchange, electrodialysis, etc.):        adding an aqueous base to the remove the Carbonates and/or        Carbamates from the DA free base mixture.

Referring to FIG. 4, some of the various combinations of steps are asfollows:

-   -   1, 2, 3, 4, 5, 6, 7, 8    -   1, 3, 4, 5, 6, 7, 8    -   1, 9, 3, 4, 5, 6, 7, 8    -   1, 2, 3, 10, 4, 5, 6, 7, 8    -   1, 3, 10, 4, 5, 6, 7, 8    -   1, 9, 3, 10, 4, 5, 6, 7, 8    -   1, 2, 3, 10, 11, 8    -   1, 3, 10, 11, 8    -   1, 9, 3, 10, 11, 8    -   1, 2, 3, 11, 8    -   1, 3, 11, 8    -   1, 9, 3, 11, 8    -   1, 2, 3, 4, 11, 8    -   1, 2, 3, 10, 4, 11, 8    -   1, 3, 10, 4, 11, 8    -   1, 3, 4, 11, 8    -   1, 9, 3, 4, 11, 8    -   1, 9, 3, 10, 4, 11, 8    -   1, 2, 3, 4, 12, 6, 7, 8    -   1, 2, 3, 10, 4, 12, 6, 7, 8    -   1, 3, 4, 12, 6, 7, 8    -   1, 3, 10, 4, 12, 6, 7, 8    -   1, 9, 3, 4, 12, 6, 7, 8    -   1, 9, 3, 10, 4, 12, 6, 7, 8    -   1, 2, 3, 4, 5, 12, 6, 7, 8    -   1, 2, 3, 10, 4, 5, 12, 6, 7, 8    -   1, 3, 4, 5, 12, 6, 7, 8    -   1, 3, 10, 4, 5, 12, 6, 7, 8    -   1, 9, 3, 4, 5, 12, 6, 7, 8    -   1, 9, 3, 10, 4, 5, 12, 6, 7, 8

The disclosed process can be applied in principle using anydiamine-containing cultured medium (e.g. HMD-containing culturedmedium). There are also in principle no limitations whatsoever regardingthe microorganisms employed in the culturing or fermentation. Themicroorganism may be naturally occurring microorganisms; microorganismsimproved by means of mutation and selection, and recombinantly producedor genetically engineered microorganisms, such as bacteria and fungi.These microorganisms are capable either of producing a DA or DAderivative, HMD and/or HMD derivatives such as HMD carbonate or HMDbicarbonate. More specifically, a recombinant organism employed iscapable of DA biosynthesis, e.g. HMD biosynthesis via the HMD pathways(“HMD pathway”) discussed below and as disclosed in U.S. Pat. No.8,377,680, or other references cited herein, which disclosures arehereby incorporated by reference in its entirety.

In some embodiments, the genetically engineered microorganism that has aDA pathway including at least one exogenous nucleic acid encoding atleast one enzyme of the DA pathway can also include an exogenous nucleicacid encoding a carbonic anhydrase enzyme. In other embodiments, thegenetically engineered microorganism has a DA synthesis pathway,preferably an HMD synthesis pathway, with at least two exogenous nucleicacids encoding at least one enzyme of the DA synthesis pathway,preferably HMD synthesis pathway, and a carbonic anhydrase enzyme orvariant expressed in a sufficient amount to produce at least one DACarbonates and/or DA Carbamates, preferably HMD Carbonates and/orCarbamates, compound. In other embodiments, the genetically engineeredmicroorganism has a DA synthesis pathway, preferably an HMD synthesispathway, with at least two exogenous nucleic acids encoding at least oneenzyme of the DA synthesis pathway, preferably the HMD synthesispathway, and a carbonic anhydrase enzyme or variant expressed in asufficient amount to produce at least one or more DA free base,preferably HMD free base, and carbon dioxide. It should be understoodthat a process for a diamine production can include the geneticallyengineered microorganisms as discussed above and that has a DA pathway,preferably HMD synthesis pathway, and a carbonic anhydrase enzyme orvariant expressed in a sufficient amount to produce at least one DACarbonates and/or DA Carbamates, preferably HMD Carbonates and/orCarbamates, compound or that can produce, at least one or more DA freebase, preferably HMD free base, and carbon dioxide or both.

Exemplary HMD synthesis pathways include pathways depicted in FIGS. 10,11, 13, 20, 21, 22, 24, 25 and 26. Disclosed are various pathways forthe production of HMD. For example, the HMD pathways include thefollowing:

-   -   a) steps depicted as A-N of FIG. 13.    -   b) steps A/L/N/C of FIG. 13.    -   c) steps M/N/C of FIG. 13.    -   d) steps D/E/F/G/H of FIG. 13).    -   e) steps D/I/J/G/H of FIG. 13).    -   f) steps D/E/K/J/G of FIG. 13    -   g) steps A-H of FIG. 15    -   h) steps A/B/C/D/E/R/S of FIG. 16    -   i) steps A/B/F/G/D/E/R/S of FIG. 16    -   j) steps A/B/H/I/D/E/R/S of FIG. 16    -   k) steps A/B/C/AB/Z/R/S of FIG. 16    -   I) steps A/B/H/I/AB/Z/R/S of FIG. 16    -   m) steps A/B/F/G/AB/Z/R/S of FIG. 16    -   n) steps A/B//J/O/P/Q/S of FIG. 16    -   o) steps A/B/J/M/N/P/Q/S of FIG. 16    -   p) steps A/B/J/K/UP/Q/S of FIG. 16    -   q) steps A/B/J/O/Z/R/S of FIG. 16    -   r) steps A/B/J/K/L/Z/R/S of FIG. 16    -   s) steps A/B/J/M/N/Z/R/S of FIG. 16    -   t) steps A/B/J/T/W/Q/S of FIG. 16    -   u) steps A/B/J/T/U/X/Q/S of FIG. 16    -   v) steps A/B/J/TN/Y/Q/S of FIG. 16    -   w) steps A-G of FIG. 17    -   x) steps O/C or D/P/G/H of FIG. 19    -   y) Steps A/B/C/G/H/I/J/K/UM of FIG. 21.    -   z) steps K/L/H of FIG. 21    -   aa) steps I/J/H of FIG. 21    -   bb); steps I/G/C of FIG. 21    -   cc) steps A/B/C of FIG. 21    -   dd) steps A/M/H of FIG. 21    -   ee) steps A/B/C of FIG. 22    -   ff) steps A/E/F/G/AA of FIG. 20

Any of the disclosed HMD synthesis pathways may be used to generate agenetically engineered microorganism that produces the pathway, pathwayintermediate or product as desired. For example, the geneticallyengineered microorganism can have a HMD pathway including at least oneexogenous nucleic acid encoding at least one enzyme of the HMD synthesispathway expressed in a sufficient amount to produce at least one HMDCarbonates and/or Carbamates compound. The genetically engineeredmicroorganism can have a HMD pathway including at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least ten, or at least eleven exogenousnucleic acids encoding enzymes of the HMD synthesis pathway. Theexogenous nucleic acids can encode, for example, polypeptides, where thepolypeptide is an enzyme or protein that can convert desired substrates,intermediates and produce products of the desired HMD synthesispathways.

In some embodiments, the genetically engineered microorganism that has aHMD pathway including at least one exogenous nucleic acid encoding atleast one enzyme of the HMD can also include an exogenous nucleic acidencoding a carbonic anhydrase enzyme.

For example, the HMD synthesis pathway may include intermediates such as3-oxoadipyl-CoA, adipate semialdehyde, 6-aminocaproate (6-ACA), 6-ACAsemialdehyde, 2-aminopimelate, 3, 6-dihydroxyhexanoyl-CoA andhomolysine.

In some embodiments, the HMD synthesis pathway may include enzymes suchas 3-oxoadipyl-CoA thiolase, 6-ACA transaminase or dehydrogenase,6-aminocaproyl-CoA reductase, 6-ACA reductase, adipyl-CoA reductase,adipate reductase, 6-hydroxy 3-oxohexanoyl-CoA dehydrogenase,2-aminopimelate decarboxylase, and homolysine decarboxylase.

In other embodiments, the HMD synthesis pathway may include an enzymeand substrate-product pair such as 3-oxoadipyl-CoA thiolase that acts onsuccinyl-CoA and acetyl-CoA to make 3-oxoadipyl-CoA, 6-ACA transaminasethat acts on adipyl-CoA to form 6-ACA, 6-aminocaproyl-CoA reductase thatacts on 6-aminocaproyl-CoA to form 6-ACA semialdehyde, 6-ACA reductasethat acts on 6-ACA and converts it directly to 6-ACA semialdehyde,adipyl-CoA reductase that acts on adipyl-CoA to form adipatesemialdehyde, adipate reductase that acts on adipate and converts itdirectly to adipate semialdehyde, 6-hydroxy 3-oxohexanoyl-CoAdehydrogenase that reduces 6-hydroxy 3-oxohexanoyl-CoA to form3,6-dihydroxy hexanoyl-CoA, 2-aminopimelate decarboxylase thatdecarboxylates 2-aminopimelate to form 6-ACA, and homolysinedecarboxylase that decarboxylates homolysine to form HMDA.

In some embodiments, a microorganism may produce the desired diamine(e.g. HMD) via a desired synthesis pathway that may include thefollowing:

-   -   (a) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase,        3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA        reductase, adipyl-CoA reductase, 6-ACA transaminase or        dehydrogenase, 6-ACA transferase or synthetase and 6-ACA-CoA        reductase, or 6-ACA reductase, HMDA transaminase or        dehydrogenase;    -   (b) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase,        3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA        reductase, adipyl-CoA reductase, 6-ACA transaminase or        dehydrogenase, 6-ACA reductase, HMDA transaminase or        dehydrogenase;    -   (c) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase,        3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA        reductase, adipyl-CoA transferase, hydrolase or transferase,        adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA        transferase or synthetase, 6-ACA-CoA reductase, HMDA        transaminase or dehydrogenase;    -   (d) 3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase,        3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA        reductase, adipyl-CoA transferase, hydrolase or transferase,        adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA        reductase, HMDA transaminase or dehydrogenase;    -   (e) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase,        3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase,        adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA        transferase or synthetase, 6-ACA-CoA reductase, HMDA        transaminase or dehydrogenase;    -   (f) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase,        3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase,        adipate reductase, 6-ACA transaminase or dehydrogenase, 6-ACA        reductase, HMDA transaminase or dehydrogenase;    -   (g) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase,        3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase,        adipyl-CoA transferase, hydrolase or transferase, adipyl-CoA        reductase, 6-ACA transaminase or dehydrogenase, 6-ACA        transferase or synthetase, 6-ACA-CoA reductase, HMDA        transaminase or dehydrogenase;    -   (h) 3-oxoadipyl-CoA thiolase, 3-oxoadipate dehydrogenase,        3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase,        adipyl-CoA transferase, hydrolase or transferase, adipyl-CoA        reductase, 6-ACA transaminase or dehydrogenase, 6-ACA reductase,        HMDA transaminase or dehydrogenase;    -   (i) an 4-hydroxy-2-oxoheptane-I,7-dioate (HODH aldolase); an        2-oxohept-4-ene-I,7-dioate (OHED) hydratase; an OHED        formate-lyase and a pyruvate formate-lyase activating enzyme or        OHED dehydrogenase; a 2,3-dehydroadipyl-CoA reductase; an        adipyl-CoA dehydrogenase; or an adipate semialdehyde        aminotransferase or an adipate semialdehyde oxidoreductase        (aminating);    -   (j) a β-ketothiolase or an acetyl-CoA carboxylase and an        acetoacetyl-CoA synthase, a 3-hydroxyacyl-CoA dehydrogenase or a        3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, and a        trans-2-enoyl-CoA reductase for producing hexanoyl-CoA, one or        more of a thioesterase, an aldehyde dehydrogenase, or a butanal        dehydrogenase, said host producing hexanal or hexanoates; one or        more of a monooxygenase, an alcohol dehydrogenase, an aldehyde        dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a        5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate        dehydrogenase, a 6-oxohexanoate dehydrogenase, or a        7-oxoheptanoate dehydrogenase, said host producing adipic acid        or adipate semialdehyde; one or more of a monooxygenase, a        transaminase, a 6-hydroxyhexanoate dehydrogenase, a        5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate        dehydrogenase, and an alcohol dehydrogenase, said host producing        6-aminohexanoate; one or more of a carboxylate reductase, a        w-transaminase, a deacetylase, a N-acetyl transferase, or an        alcohol dehydrogenase. Such pathway is disclosed in U.S. Patent        Application Publication No. 20140186902;    -   (k) acetyltransferase or thiolase to form        6-hydroxy-3-oxo-hexanoyl-CoA, 6-hydroxy-3-oxo-hexanoyl-CoA        dehydrogenase, 3,4-dihydroxyhexanoyl-CoA dehydratase,        6-hydroxy-2-hexenoyl-CoA reductase, 6-hydroxyhexanoyl-CoA        hydrolase to form 6-ACA, 6-hydroxycaproate dehydrogenase and        transaminase to form HMDA. Such pathway is disclosed in        International Application Publication No. WO 2014/047407A1;    -   (l) homocitrate synthase, a homoaconitase and a homoisocitrate        dehydrogenase to form 2-ketopimelate, 2-keto decarboxylase        catalyzing the conversion of α-ketopimelate to adipate        semialdehyde, 2-aminotransferase catalyzes the conversion of        α-ketopimelate to 2-aminopimelate, 2-aminopimelate decarboxylase        to decarboxylate 2-aminopimelate and form 6-ACA, aldehyde        dehydrogenase catalyzes the conversion of 6-ACA to        6-aminohexanal and the aminotransferase catalyzes the conversion        of 6-aminohexanal to 6-hexamethylenediamine. Such pathway is        disclosed in International Application Publication No. in        WO/2010/068944; and    -   (m) glutamyl-CoA transferase and/or ligase, beta-ketothiolase,        3-oxo-6-aminopimeloyl-CoA oxidoreductase,        3-hydroxy-6-aminopimeloyl-CoA dehydratase,        6-amino-7-carboxyhept-2-enoyl-CoA reductase, 6-aminopimeloyl-CoA        reductase (aldehyde forming), 2-amino-7-oxoheptanoate        aminotransferase and/or aminating oxidoreductase, homolysine        decarboxylase, 6-aminopimeloyl-CoA hydrolase, transferase and/or        ligase, 2-aminopimelate decarboxylase. Such pathway is disclosed        in International Application Publication No. WO 2010/129936

In some embodiments, the HMD synthesis pathway includes at least oneenzyme and the nucleic acids encoding such one or more enzymes for3-oxoadipyl-CoA thiolase, 3-oxoadipyl-CoA dehydrogenase,3-hydroxyadipyl-CoA dehydratase, 5-carboxy-2-pentenoyl-CoA reductase,adipyl-CoA reductase, 6-ACA transaminase or dehydrogenase,3-oxoadipyl-CoA:acyl CoA transferase, 3-oxoadipate dehydrogenase,3-hydroxyadipate dehydratase, 5-carboxy-2-pentenoate reductase,adipyl-CoA transferase, ligase, or hydrolase, 6-ACA transferase orsynthetase, 6-ACA-CoA reductase, HMDA transaminase or dehydrogenase,adipate reductase, 6-ACA transaminase or dehydrogenase, or 6-ACAreductase.

Suitable microorganisms that can be used as a host to include one ormore exogenous nucleic acids of the HMD synthesis pathways include forexample prokaryotes such as bacteria, and eukaryotes such as, fungus(e.g. yeast) or any other microorganisms applicable to fermentationprocesses or that can tolerate pH conditions below pH 4.

In some embodiments, the genetically engineered microorganismEscherichia, Klebsiella; the order Aeromonadales, familySuccinivibrionaceae, including the genus Anaerobiospirillum; the orderPasteurellales, family Pasteurellaceae, including the generaActinobacillus and Mannheimia; the order Rhizobiales, familyBradyrhizobiaceae, including the genus Rhizobium; the order Bacillales,family Bacillaceae, including the genus Bacillus; the orderActinomycetales, families Corynebacteriaceae and Streptomycetaceae,including the genus Corynebacterium and the genus Streptomyces,respectively; order Rhodospirillales, family Acetobacteraceae, includingthe genus Gluconobacter; the order Sphingomonadales, familySphingomonadaceae, including the genus Zymomonas; the orderLactobacillales, families Lactobacillaceae and Streptococcaceae,including the genus Lactobacillus and the genus Lactococcus,respectively; the order Clostridiales, family Clostridiaceae, genusClostridium; and the order Pseudomonadales, family Pseudomonadaceae,including the genus Pseudomonas, the genus Alkaliphilus,Methylobacterium, Methyloversatilis, Methylococcus, Methylocystis andHyphomicrobium the order Saccharomycetales, family Saccaromycetaceae,including the genera Saccharomyces, Kluyveromyces and Pichia; the orderSaccharomycetales, family Dipodascaceae, including the genus Yarrowia;the order Schizosaccharomycetales, family Schizosaccaromycetaceae,including the genus Schizosaccharomyces; the order Eurotiales, familyTrichocomaceae, including the genus Aspergillus; and the orderMucorales, family Mucoraceae, including the genus Rhizopus.

In other embodiments the genetically engineered microorganism comprisesnon-limiting species of host bacteria include Escherichia coli,Klebsiella oxytoca, Anaerobiospirillum succiniciproducens,Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobiumetli, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonasmobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomycescoelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, andPseudomonas putida, Bacillis pseudofirmus, Bacillus halodurans, Bacillusalcalophilus, Clostridium paradoxum, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Hansenula polymorpha, Pichia methanolica,Candida boidinii, Kluyveromyces lactis, Kluyveromyces marxianus,Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopusarrhizus, Rhizopus oryzae, Yarrowia lipolytica, and Issatchenkiaorientalis. Some alkaliphiles are: Bacillis pseudofirmus, Bacillushalodurans, Bacillus alcalophilus, Clostridium paradoxum, Arthrospiraplatensis, Bacillus clausii, Oceanobacillus iheyensis, Alkaliphilusmetalliredigens, Alkaliphilus oremlandii, Bacillus selentireducens,Desulfovibrio alkaliphiles, Dethiobacter alkaliphiles, Thioalkalivibriosp., Natranaerobius thermophilus, Alkalilimnicola ehrlichii, andDesulfonatronospira thiodismutans.

In some embodiments, the species of fungi or yeast can be selected fromSaccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenulapolymorpha, Pichia methanolica, Candida boidinii, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichiapastoris, Rhizopus arrhizus, Rhizopus oryzae, Yarrowia lipolytica, andIssatchenkia orientalis and the like.

In some embodiments, the genetically engineered microorganism isEscherichia coli, Corynebacterium glutamicum, Bacillus subtilis,Pseudomonas putida, Bacillis pseudofirmus, Bacillus halodurans, Bacillusalcalophilus, Clostridium paradoxum, Saccharomyces cerevisiae.

For example, E. coli is a particularly useful host organism since it isa well characterized microbial organism suitable for geneticengineering. Other particularly useful host organisms include yeast suchas Saccharomyces cerevisiae. In some embodiments, genetically engineeredmicroorganisms are modified that have improved alkali tolerance.

Alkali tolerance can be introduced into organisms that are not typicallyalkali-tolerant using adaptive evolution. Cells are grown underincreasingly higher pH conditions till they have adapted their cellularmechanisms to grow optimally at the desired high pH (alkaline pH).Typically, small volumes of cells, still growing in exponential phase,are transferred to a fresh medium at a predetermined pH till they havereached a certain biomass concentration. These cells are then dilutedinto a fresh medium where an incrementally higher pH is maintained. Thisprocess selects for cells that are more fit to grow at the higher pH.The process of transferring small volume of exponentially growing cellsinto fresh medium at higher pH is continued till the cells have evolvedto grow at the target pH level.

Adaptive evolution has been used to evolve strains to grow onnon-natural substrates (Lee and Palsson, Appl Environ Microbiol. 2010July; 76(13):4158-68, Adaptive evolution of Escherichia coli K-12 MG1655during growth on a Nonnative carbon source, L-1,2-propanediol), forimproved salt tolerance (Ketola and Hiltunen, Ecol Evol. 2014 October;4:3901-8, Rapid evolutionary adaptation to elevated salt concentrationsin pathogenic freshwater bacteria Serratia marcescens), for improvedproduct tolerance (Kildegaard K R et al., Metab Eng. 2014 Sep. 28;26C:57-66, Evolution reveals a glutathione-dependent mechanism of3-hydroxypropionic acid tolerance), for growth at high temperature(Sandeberg et al., Mol Biol Evol. 2014 October; 31(10):2647-62,Evolution of Escherichia coli to 42° C. and subsequent geneticengineering reveals adaptive mechanisms and novel mutations), to evolvefor fermentation under aerobic conditions (Portnoy et al., Appl EnvironMicrobiol. 2008 December; 74(24), Aerobic fermentation of D-glucose byan evolved cytochrome oxidase-deficient Escherichia coli strain) amongseveral other objectives.

For example, one of the pathways entails the activation of6-aminocaproate to 6-aminocaproyl-CoA by a transferase or synthaseenzyme (FIG. 10, Step Q or R) followed by the spontaneous cyclization of6-aminocaproyl-CoA to form caprolactam (FIG. 10, Step T). In otherdescribed pathways, the pathway entails the activation of6-aminocaproate to 6-aminocaproyl-CoA (FIG. 10, Step Q or R), followedby a reduction (FIG. 10, Step U) and amination (FIG. 10, Step V or W) toform HMD. 6-Aminocaproic acid can alternatively be activated to6-aminocaproyl-phosphate instead of 6-aminocaproyl-CoA.6-Aminocaproyl-phosphate can spontaneously cyclize to form caprolactam.Alternatively, 6-aminocaproyl-phosphate can be reduced to6-aminocaproate semialdehye, which can be then converted to HMD asdepicted in FIGS. 10 and 11. In either case, the amination reaction mustoccur relatively quickly to minimize the spontaneous formation of thecyclic imine of 6-aminocaproate semialdehyde. Linking or scaffolding theparticipating enzymes represents a potentially powerful option forensuring that the 6-aminocaproate semialdehyde intermediate isefficiently channeled from the reductase enzyme to the amination enzyme.

Another option for minimizing or even eliminating the formation of thecyclic imine or caprolactam during the conversion of 6-aminocaproic acidto HMD entails adding a functional group (for example, acetyl, succinyl)to the amine group of 6-aminocaproic acid to protect it fromcyclization. This is analogous to ornithine formation from L-glutamatein Escherichia coli. Specifically, glutamate is first converted toN-acetyl-L-glutamate by N-acetylglutamate synthase. N-Acetyl-L-glutamateis then activated to N-acetylglutamyl-phosphate, which is reduced andtransaminated to form N-acetyl-L-ornithine. The acetyl group is thenremoved from N-acetyl-L-ornithine by N-acetyl-L-ornithine deacetylaseforming L-ornithine. Such a route is necessary because formation ofglutamate-5-phosphate from glutamate followed by reduction toglutamate-5-semialdehyde leads to the formation of(S)-1-pyrroline-5-carboxylate, a cyclic imine formed spontaneously fromglutamate-5-semialdehyde. In the case of forming HMD from 6-aminocaproicacid, the steps can involve acetylating 6-aminocaproic acid toacetyl-6-aminocaproic acid, activating the carboxylic acid group with aCoA or phosphate group, reducing, aminating, and deacetylating.

Note that 6-aminocaproate can be formed from various starting molecules.For example, the carbon backbone of 6-aminocaproate can be derived fromsuccinyl-CoA and acetyl-CoA as depicted in FIG. 10 and also described inFIGS. 2, 3 and 8. Alternatively, 6-aminocaproate can be derived fromalpha-ketoadipate, where alpha-ketoadipate is converted to adipyl-CoA(see FIG. 9), and adipyl-CoA is converted to 6-aminocaproate as shown inFIG. 10.

FIG. 11 provides two additional metabolic pathways to 6-aminocaproate or6-aminocapropyl-CoA starting from 4-aminobutyryl-CoA and acetyl-CoA. Thefirst route entails the condensation of 4-aminobutyryl-CoA andacetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA (Step A) followed by areduction (Step B), dehydration (Step C), and reduction (Step D) to form6-aminocaproyl-CoA. 6-Aminocaproyl-CoA can be converted to6-aminocaproate by a transferase (Step K), synthase (Step L), orhydrolase (Step M) enzyme. Alternatively, 6-aminocaproyl-CoA can beconverted to caprolactam by spontaneous cyclization (Step Q) or to HMDfollowing its reduction (Step N) and amination (Step O or P). The secondpathway described in FIG. 11 entails the condensation of4-aminobutyryl-CoA and acetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA(Step A) which is then converted to 3-oxo-6-aminohexanoate by atransferase (Step E), synthase (Step F), or hydrolase (Step G).3-Oxo-6-aminohexanoate is then reduced (Step H), dehydrated (Step I),and reduced (Step J) to form 6-aminocaproate.

The starting molecule, 4-aminobutyryl-CoA, can be formed from variouscommon central metabolites. For example, glutamate can be decarboxylatedto 4-aminobutyrate, which is then activated by a CoA-transferase orsynthase to 4-aminobutyryl-CoA. Alternatively, succinate semialdehyde,formed from either the reduction of succinyl-CoA or the decarboxylationof alpha-ketoglutarate, can be transaminated to 4-aminobutyrate prior toactivation by a CoA-transferase or synthase to form 4-aminobutyryl-CoA.It is noted that 4-aminobutyryl-CoA and several of the intermediates ofthe 4-aminobutyryl-CoA to 6-aminocaproyl-CoA pathway may spontaneouslycyclize to their corresponding lactams. Thus, adding a protectivefunctional group to the terminal amine group of 4-aminobutyryl-CoAand/or several of the amino-CoA intermediates can be used to minimizethe formation of unwanted cyclic byproducts. In this case, the samegeneral set of transformations depicted in FIG. 11 would apply, althoughtwo additional steps, for example, an acetylase and deacetylase, can beadded to the pathway.

All transformations depicted in FIGS. 10-11 fall into the 12 generalcategories of transformations shown in Table 8. Below is described anumber of biochemically characterized candidate genes in each category.Specifically listed are genes that can be applied to catalyze theappropriate transformations in FIGS. 10-11 when cloned and expressed.

TABLE 8 Enzyme types for conversion of succinyl-CoA, acetyl-CoA, and/or4-aminobutyryl-CoA to 6-amino-caproate, caprolactam, and/orhexamethylenediamine. The first three digits of each label correspond tothe first three Enzyme Commission number digits which denote the generaltype of transformation independent of substrate specificity. LabelFunction 1.1.1.a Oxidoreductase (ketone to hydroxyl or aldehyde toalcohol) 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) 1.3.1.aOxidoreductase operating on CH—CH donors 1.4.1.a Oxidoreductaseoperating on amino acids 2.3.1.b Acyltransferase 2.6.1.aAminotransferase 2.8.3.a Coenzyme-A transferase 3.1.2.a Thiolesterhydrolase (CoA specific) 4.2.1.a Hydro-lyase 6.2.1.a Acid-thiol ligase6.3.1.a/6.3.2.a Amide synthases/peptide synthases No enzyme Spontaneouscyclization required

1.1.1.a Oxidoreductases. Four transformations depicted in FIGS. 10 and11 require oxidoreductases that convert a ketone functionality to ahydroxyl group. Step B in both FIGS. 10 and 11 involves converting a3-oxoacyl-CoA to a 3-hydroxyacyl-CoA. Step H in both FIGS. 1 and 2involves converting a 3-oxoacid to a 3-hydroxyacid.

Exemplary enzymes that can convert 3-oxoacyl-CoA molecules such as3-oxoadipyl-CoA and 3-oxo-6-aminohexanoyl-CoA into 3-hydroxyacyl-CoAmolecules such as 3-hydroxyadipyl-CoA and 3-hydroxy-6-aminohexanoyl-CoA,respectively, include enzymes whose natural physiological roles are infatty acid beta-oxidation or phenylacetate catabolism. For example,subunits of two fatty acid oxidation complexes in E. coli, encoded byfadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock etal., Methods Enzymol. 71:403-411 (1981)). Furthermore, the gene productsencoded by phaC in Pseudomonas putida U (Olivera et al., Proc. Natl.Acad. Sci. USA 95:6419-6424 (1998)) and paaC in Pseudomonas fluorescensST (Di Gennaro et al., Arch. Microbiol. 188:117-125 (2007)) catalyze thereverse reaction of step B in FIG. 10, that is, the oxidation of3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during the catabolism ofphenylacetate or styrene. Note that the reactions catalyzed by suchenzymes are reversible. In addition, given the proximity in E. coli ofpaaH to other genes in the phenylacetate degradation operon (Nogales etal., Microbiology 153:357-365 (2007)) and the fact that paaH mutantscannot grow on phenylacetate (Ismail et al., Eur. J. Biochem.270:3047-3054 (2003)), it is expected that the E. coli paaH gene encodesa 3-hydroxyacyl-CoA dehydrogenase.

Gene GenBank name GI# Accession # Organism fadB 119811 P21177.2Escherichia coli fadJ 3334437 P77399.1 Escherichia coli paaH 16129356NP_415913.1 Escherichia coli phaC 26990000 NP_745425.1 Pseudomonasputida paaC 106636095 ABF82235.1 Pseudomonas fluorescens

Additional exemplary oxidoreductases capable of converting 3-oxoacyl-CoAmolecules to their corresponding 3-hydroxyacyl-CoA molecules include3-hydroxybutyryl-CoA dehydrogenases. The enzyme from Clostridiumacetobutylicum, encoded by hbd, has been cloned and functionallyexpressed in E. coli (Youngleson et al., J. Bacteriol. 171:6800-6807(1989)). Additional gene candidates include Hbd1 (C-terminal domain) andHbd2 (N-terminal domain) in Clostridium kluyveri (Hillmer et al., FEBSLett. 21:351-354 (1972)) and HSD17B10 in Bos taurus (Wakil et al., J.Biol. Chem. 207:631-638 (1954)). Yet other gene candidates demonstratedto reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloearamigera (Ploux et al., Eur. J. Biochem. 174:177-182 (1988)) and phaBfrom Rhodobacter sphaeroides (Alber et al., Mol. Microbiol 61:297-309(2006)). The former gene candidate is NADPH-dependent, its nucleotidesequence has been determined (Peoples et al., Mol. Microbiol. 3:349-357(1989)) and the gene has been expressed in E. coli. Substratespecificity studies on the gene led to the conclusion that it couldaccept 3-oxopropionyl-CoA as a substrate besides acetoacetyl-CoA (Plouxet al., supra).

GenBank Gene name GI# Accession # Organism hbd 18266893 P52041.2Clostridium acetobutylicum Hbd2 146348271 EDK34807.1 Clostridiumkluyveri Hbd1 146345976 EDK32512.1 Clostridium kluyveri HSD17B10 3183024O02691.3 Bos taurus phbB 130017 P23238.1 Zoogloea ramigera phaB146278501 YP_001168660.1 Rhodobacter sphaeroides

A number of similar enzymes have been found in other species ofClostridia and in Metallosphaera sedula (Berg et al., Science318:1782-1786 (2007)).

GenBank Gene name GI# Accession # Organism hbd 15895965 NP_349314.1Clostridium acetobutylicum hbd 20162442 AAM14586.1 Clostridiumbeijerinckii Msed_1423 146304189 YP_001191505 Metallosphaera sedulaMsed_0399 146303184 YP_001190500 Metallosphaera sedula Msed_0389146303174 YP_001190490 Metallosphaera sedula Msed_1993 146304741YP_001192057 Metallosphaera sedula

Various alcohol dehydrogenases represent good candidates for converting3-oxoadipate to 3-hydroxyadipate (step H, FIG. 10) or3-oxo-6-aminohexanoate to 3-hydroxy-6-aminohexanoate (step H, FIG. 11).Two such enzymes capable of converting an oxoacid to a hydroxyacid areencoded by the malate dehydrogenase (mdh) and lactate dehydrogenase(IdhA) genes in E. coli. In addition, lactate dehydrogenase fromRalstonia eutropha has been shown to demonstrate high activities onsubstrates of various chain lengths such as lactate, 2-oxobutyrate,2-oxopentanoate and 2-oxoglutarate (Steinbuchel et al., Eur. J. Biochem.130:329-334 (1983)). Conversion of alpha-ketoadipate intoalpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, anenzyme reported to be found in rat and in human placenta (Suda et al.,Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem.Biophys. Res. Commun. 77:586-591 (1977)). An additional candidate forthese steps is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh)from the human heart which has been cloned and characterized (Marks etal., J. Biol. Chem. 267:15459-15463 (1992)). This enzyme is adehydrogenase that operates on a 3-hydroxyacid. Another exemplaryalcohol dehydrogenase converts acetone to isopropanol as was shown in C.beijerinckii (Ismaiel et al., J. Bacteriol. 175:5097-5105 (1993) and T.brockii (Lamed et al., Biochem. J. 195:183-190 (1981); Peretz et al.,Biochemistry 28:6549-6555 (1989)).

GenBank Gene name GI# Accession # Organism mdh 1789632 AAC76268.1Escherichia coli ldhA 16129341 NP_415898.1 Escherichia coli ldh113866693 YP_725182.1 Ralstonia eutropha bdh 177198 AAA58352.1 Homosapiens adh 60592974 AAA23199.2 Clostridium beijerinckii adh 113443P14941.1 Thermoanaerobacter brockii

1.2.1.b Oxidoreductase (acyl-CoA to aldehyde). The transformations ofadipyl-CoA to adipate semialdehyde (Step N, FIGS. 10) and6-aminocaproyl-CoA to 6-aminocaproate semialdehyde (Step U, FIG. 10;Step N, FIG. 11) require acyl-CoA dehydrogenases capable of reducing anacyl-CoA to its corresponding aldehyde. Exemplary genes that encode suchenzymes include the Acinetobacter calcoaceticus acrl encoding a fattyacyl-CoA reductase (Reiser et al, J. Bacteriology 179:2969-2975 (1997)),the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl.Environ. Microbiol. 68:1192-1195 (2002)), and a CoA- and NADP-dependentsuccinate semialdehyde dehydrogenase encoded by the sucD gene inClostridium kluyveri (Sohling et al., J. Bacteriol. 178:871-880 (1996)).SucD of P. gingivalis is another succinate semialdehyde dehydrogenase(Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). The enzymeacylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG,is yet another candidate as it has been demonstrated to oxidize andacylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehydeand formaldehyde (Powlowski et al., J Bacteriol. 175:377-385 (1993)). Inaddition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhEin Leuconostoc mesenteroides has been shown to oxidize the branchedchain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J.Gen. Appl. Microbiol. 18:43-55 (1972); Koo et al., Biotechnol Lett.27:505-510 (2005)).

GenBank Gene name GI# Accession # Organism acr1 50086359 YP_047869.1Acinetobacter calcoaceticus acr1 1684886 AAC45217 Acinetobacter baylyiacr1 18857901 BAB85476.1 Acinetobacter sp. Strain M-1 sucD 172046062P38947.1 Clostridium kluyveri sucD 34540484 NP_904963.1 Porphyromonasgingivalis bphG 425213 BAA03892.1 Pseudomonas sp adhE 55818563AAV66076.1 Leuconostoc mesenteroides

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archaeal bacteria (Berg et al., supra; Thauer R. K.,Science 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactorand has been characterized in Metallosphaera and Sulfolobus spp (Alberet al., J. Bacteriol. 188:8551-8559 (2006); Hugler et al., J. Bacteriol.184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 inMetallosphaera sedula (Alber et al., supra; Berg et al., supra). A geneencoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned andheterologously expressed in E. coli (Alber et al., supra). This enzymehas also been shown to catalyze the conversion of methylmalonyl-CoA toits corresponding aldehyde (WO/2007/141208). Although the aldehydedehydrogenase functionality of these enzymes is similar to thebifunctional dehydrogenase from Chloroflexus aurantiacus, there islittle sequence similarity. Both malonyl-CoA reductase enzyme candidateshave high sequence similarity to aspartate-semialdehyde dehydrogenase,an enzyme catalyzing the reduction and concurrent dephosphorylation ofaspartyl-4-phosphate to aspartate semialdehyde. Additional genecandidates can be found by sequence homology to proteins in otherorganisms including Sulfolobus solfataricus and Sulfolobusacidocaldarius and have been listed below. Yet another candidate forCoA-acylating aldehyde dehydrogenase is the ald gene from Clostridiumbeijerinckii (Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)).This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA totheir corresponding aldehydes. This gene is very similar to eutE thatencodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli(Toth et al., supra).

Gene name GI# GenBank Accession # Organism Msed_0709 146303492YP_001190808.1 Metallosphaera sedula mcr 15922498 NP_378167.1 Sulfolobustokodaii asd-2 15898958 NP_343563.1 Sulfolobus solfataricus Saci_237070608071 YP_256941.1 Sulfolobus acidocaldarius Ald 49473535 AAT66436Clostridium beijerinckii eutE 687645 AAA80209 Salmonella typhimuriumeutE 2498347 P77445 Escherichia coli

1.3.1.a Oxidoreductase operating on CH—CH donors. Referring to FIG. 10,step D refers to the conversion of 5-carboxy-2-pentenoyl-CoA toadipyl-CoA by 5-carboxy-2-pentenoyl-CoA reductase. Referring to FIG. 11,step D refers to the conversion of 6-aminohex-2-enoyl-CoA to6-aminocaproyl-CoA. Enoyl-CoA reductase enzymes are suitable enzymes foreither transformation. One exemplary enoyl-CoA reductase is the geneproduct of bcd from C. acetobutylicum (Boynton et al., J Bacteriol.178:3015-3024 (1996); Atsumi et al., Metab. Eng. 2008 10(6):305-311(2008)(Epub Sep. 14, 2007), which naturally catalyzes the reduction ofcrotonyl-CoA to butyryl-CoA. Activity of this enzyme can be enhanced byexpressing bcd in conjunction with expression of the C. acetobutylicumetfAB genes, which encode an electron transfer flavoprotein. Anadditional candidate for the enoyl-CoA reductase step is themitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et al.,J. Biol. Chem. 280:4329-4338 (2005)). A construct derived from thissequence following the removal of its mitochondrial targeting leadersequence was cloned in E. coli resulting in an active enzyme(Hoffmeister et al., supra). This approach is well known to thoseskilled in the art of expressing eukaryotic genes, particularly thosewith leader sequences that may target the gene product to a specificintracellular compartment, in prokaryotic organisms. A close homolog ofthis gene, TDE0597, from the prokaryote Treponema denticola represents athird enoyl-CoA reductase which has been cloned and expressed in E. coli(Tucci et al., FEBS Letters 581:1561-1566 (2007)).

GenBank Gene name GI# Accession # Organism bcd 15895968 NP_349317.1Clostridium acetobutylicum etfA 15895966 NP_349315.1 Clostridiumacetobutylicum etfB 15895967 NP_349316.1 Clostridium acetobutylicum TER62287512 Q5EU90.1 Euglena gracilis TDE0597 42526113 NP_971211.1Treponema denticola

Step J of both FIGS. 10 and 11 requires a 2-enoate reductase enzyme.2-Enoate reductases (EC 1.3.1.31) are known to catalyze theNAD(P)H-dependent reduction of a wide variety of α,β-unsaturatedcarboxylic acids and aldehydes (Rohdich et al., J. Biol. Chem.276:5779-5787 (2001)). 2-Enoate reductase is encoded by enr in severalspecies of Clostridia (Giese) et al., Arch Microbiol 135:51-57 (1983))including C. tyrobutyricum, and C. thermoaceticum (now called Moorellathermoaceticum) (Rohdich et al., supra). In the published genomesequence of C. kluyveri, 9 coding sequences for enoate reductases havebeen reported, out of which one has been characterized (Seedorf et al.,Proc. Natl. Acad. Sci. USA, 105:2128-2133 (2008)). The enr genes fromboth C. tyrobutyricum and C. thermoaceticum have been cloned andsequenced and show 59% identity to each other. The former gene is alsofound to have approximately 75% similarity to the characterized gene inC. kluyveri (Giese) et al., supra). It has been reported based on thesesequence results that enr is very similar to the dienoyl CoA reductasein E. coli (fadH) (Rohdich et al., supra). The C. thermoaceticum enrgene has also been expressed in an enzymatically active form in E. coli(Rohdich et al., supra).

Gene name GI# GenBank Accession # Organism fadH 16130976 NP_417552.1Escherichia coli enr 169405742 ACA54153.1 Clostridium botulinum A3 strenr 2765041 CAA71086.1 Clostridium tyrobutyricum enr 3402834 CAA76083.1Clostridium kluyveri enr 83590886 YR_430895.1 Moorella thermoacetica

1.4.1.a Oxidoreductase operating on amino acids. FIG. 10 depicts tworeductive aminations. Specifically, step P of FIG. 10 involves theconversion of adipate semialdehyde to 6-aminocaproate and step W of FIG.10 entails the conversion of 6-aminocaproate semialdehyde tohexamethylenediamine. The latter transformation is also required in FIG.11, Step P.

Most oxidoreductases operating on amino acids catalyze the oxidativedeamination of alpha-amino acids with NAD+ or NADP+ as acceptor, thoughthe reactions are typically reversible. Exemplary oxidoreductasesoperating on amino acids include glutamate dehydrogenase (deaminating),encoded by gdhA, leucine dehydrogenase (deaminating), encoded by ldh,and aspartate dehydrogenase (deaminating), encoded by nadX. The gdhAgene product from Escherichia coli (McPherson et al., Nucleic. AcidsRes. 11:5257-5266 (1983); Korber et al., J. Mol. Biol. 234:1270-1273(1993)), gdh from Thermotoga maritima (Kort et al., Extremophiles1:52-60 (1997); Lebbink et al., J. Mol. Biol. 280:287-296 (1998);Lebbink et al., J. Mol. Biol. 289:357-369 (1999)), and gdhA1 fromHalobacterium salinarum (Ingoldsby et al., Gene. 349:237-244 (2005))catalyze the reversible interconversion of glutamate to 2-oxoglutarateand ammonia, while favoring NADP(H), NAD(H), or both, respectively. Theldh gene of Bacillus cereus encodes the LeuDH protein that has a wide ofrange of substrates including leucine, isoleucine, valine, and2-aminobutanoate (Stoyan et al., J. Biotechnol 54:77-80 (1997); Ansorgeet al., Biotechnol Bioeng. 68:557-562 (2000)). The nadX gene fromThermotoga maritime encoding for the aspartate dehydrogenase is involvedin the biosynthesis of NAD (Yang et al., J. Biol. Chem. 278:8804-8808(2003)).

Gene name GI# Accession # Organism gdhA 118547 P00370 Escherichia coligdh 6226595 P96110.4 Thermotoga maritima gdhA1 15789827 NP_279651.1Halobacterium salinarum ldh 61222614 P0A393 Bacillus cereus nadX15644391 NP_229443.1 Thermotoga maritima

The lysine 6-dehydrogenase (deaminating), encoded by the lysDH genes,catalyze the oxidative deamination of the ε-amino group of L-lysine toform 2-aminoadipate-6-semialdehyde, which in turn nonenzymaticallycyclizes to form Δ¹-piperideine-6-carboxylate (Misono et al., J.Bacteriol. 150:398-401 (1982)). Exemplary enzymes can be found inGeobacillus stearothermophilus (Heydari et al., Appl Environ. Microbiol70:937-942 (2004)), Agrobacterium tumefaciens (Hashimoto et al., JBiochem 106:76-80 (1989); Misono et al., supra), and Achromobacterdenitrificans (Ruldeekulthamrong et al., BMB. Rep. 41:790-795 (2008)).Such enzymes are particularly good candidates for converting adipatesemialdehyde to 6-aminocaproate given the structural similarity betweenadipate semialdehyde and 2-aminoadipate-6-semialdehyde.

GenBank Gene name GI# Accession # Organism lysDH 13429872 BAB39707Geobacillus stearothermophilus lysDH 15888285 NP_353966 Agrobacteriumtumefaciens lysDH 74026644 AAZ94428 Achromobacter denitrificans

2.3.1.b Acyl transferase. Referring to FIG. 10, step A involves3-oxoadipyl-CoA thiolase, or equivalently, succinyl CoA:acetyl CoA acyltransferase (β-ketothiolase). The gene products encoded by pcaF inPseudomonas strain B13 (Kaschabek et al., J. Bacteriol. 184:207-215(2002)), phaD in Pseudomonas putida U (Olivera et al., supra), paaE inPseudomonas fluorescens ST (Di Gennaro et al., supra), and paaJ from E.coli (Nogales et al., supra) catalyze the conversion of 3-oxoadipyl-CoAinto succinyl-CoA and acetyl-CoA during the degradation of aromaticcompounds such as phenylacetate or styrene. Since β-ketothiolase enzymescatalyze reversible transformations, these enzymes can be employed forthe synthesis of 3-oxoadipyl-CoA. For example, the ketothiolase phaAfrom R. eutropha combines two molecules of acetyl-CoA to formacetoacetyl-CoA (Sato et al., J Biosci Bioeng 103:38-44 (2007)).Similarly, a 3-keto thiolase (bktB) has been reported to catalyze thecondensation of acetyl-CoA and propionyl-CoA to form β-ketovaleryl-CoA(Slater et al., J. Bacteriol. 180:1979-1987 (1998)) in R. eutropha. Inaddition to the likelihood of possessing 3-oxoadipyl-CoA thiolaseactivity, all such enzymes represent good candidates for condensing4-aminobutyryl-CoA and acetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA(step A, FIG. 11) either in their native forms or once they have beenappropriately engineered.

Gene name GI# GenBank Accession # Organism paaJ 16129358 NP_415915.1Escherichia coli pcaF 17736947 AAL02407 Pseudomonas knackmussii (B13)phaD 3253200 AAC24332.1 Pseudomonas putida paaE 106636097 ABF82237.1Pseudomonas fluorescens

2-Amino-4-oxopentanoate (AKP) thiolase or AKP thiolase (AKPT) enzymespresent additional candidates for performing step A in FIGS. 10 and 11.AKPT is a pyridoxal phosphate-dependent enzyme participating inornithine degradation in Clostridium sticklandii (Jeng et al.,Biochemistry 13:2898-2903 (1974); Kenklies et al., Microbiology145:819-826 (1999)). A gene cluster encoding the alpha and beta subunitsof AKPT (or -2 (ortA) and or -3 (ortB)) was recently identified and thebiochemical properties of the enzyme were characterized (Fonknechten etal., J. Bacteriol. In Press (2009)). The enzyme is capable of operatingin both directions and naturally reacts with the D-isomer of alanine.AKPT from Clostridium sticklandii has been characterized but its proteinsequence has not yet been published. Enzymes with high sequence homologyare found in Clostridium difficile, Alkaliphilus metalliredigenes QYF,Thermoanaerobacter sp. X514, and Thermoanaerobacter tengcongensis MB4(Fonknechten et al., supra).

Gene name GI# GenBank Accession # Organism ortA (α) 126698017YP_001086914.1 Clostridium difficile 630 ortB (β) 126698018YP_001086915.1 Clostridium difficile 630 Amet_2368 (α) 150390132YP_001320181.1 Alkaliphilus metalliredigenes QYF Amet_2369 (β) 150390133YP_001320182.1 Alkaliphilus metalliredigenes QYF Teth514_1478 167040116YP_001663101.1 Thermoanaerobacter sp. X514 (α) Teth514_1479 167040117YP_001663102.1 Thermoanaerobacter sp. X514 (β) TTE1235 (α) 20807687NP_622858.1 Thermoanaerobacter tengcongensis MB4 thrC (β) 20807688NP_622859.1 Thermoanaerobacter tengcongensis MB4

2.6.1.a Aminotransferase. Step O of FIGS. 10 and 11 and Step V of FIG.10 require transamination of a 6-aldehyde to an amine. Thesetransformations can be catalyzed by gamma-aminobutyrate transaminase(GABA transaminase). One E. coli GABA transaminase is encoded by gabTand transfers an amino group from glutamate to the terminal aldehyde ofsuccinyl semialdehyde (Bartsch et al., J. Bacteriol. 172:7035-7042(1990)). The gene product of puuE catalyzes another 4-aminobutyratetransaminase in E. coli (Kurihara et al., J. Biol. Chem. 280:4602-4608(2005)). GABA transaminases in Mus musculus, Pseudomonas fluorescens,and Sus scrofa have been shown to react with 6-aminocaproic acid(Cooper, Methods Enzymol. 113:80-82 (1985); Scott et al., J. Biol. Chem.234:932-936 (1959)).

Gene name GI# GenBank Accession # Organism gabT 16130576 NP_417148.1Escherichia coli puuE 16129263 NP_415818.1 Escherichia coli abat37202121 NP_766549.2 Mus musculus gabT 70733692 YP_257332.1 Pseudomonasfluorescens abat 47523600 NP_999428.1 Sus scrofa

Additional enzyme candidates include putrescine aminotransferases orother diamine aminotransferases. Such enzymes are particularly wellsuited for carrying out the conversion of 6-aminocaproate semialdehydeto hexamethylenediamine. The E. coli putrescine aminotransferase isencoded by the ygjG gene and the purified enzyme also was able totransaminate cadaverine and spermidine (Samsonova et al., BMC Microbiol3:2 (2003)). In addition, activity of this enzyme on 1,7-diaminoheptaneand with amino acceptors other than 2-oxoglutarate (e.g., pyruvate,2-oxobutanoate) has been reported (Samsonova et al., supra; Kim, K. H.,J Biol Chem 239:783-786 (1964)). A putrescine aminotransferase withhigher activity with pyruvate as the amino acceptor thanalpha-ketoglutarate is the spuC gene of Pseudomonas aeruginosa (Lu etal., J Bacteriol 184:3765-3773 (2002)).

Gene name GI# GenBank Accession # Organism ygjG 145698310 NP_417544Escherichia coli spuC 9946143 AAG03688 Pseudomonas aeruginosa1. Yet additional candidate enzymes includebeta-alanine/alpha-ketoglutarate aminotransferases which producemalonate semialdehyde from beta-alanine (WO08027742). The gene productof SkPYD4 in Saccharomyces kluyveri was also shown to preferentially usebeta-alanine as the amino group donor (Andersen et al., FEBS. J.274:1804-1817 (2007)). SkUGA1 encodes a homologue of Saccharomycescerevisiae GABA aminotransferase, UGA1 (Ramos et al., Eur. J. Biochem.,149:401-404 (1985)), whereas SkPYD4 encodes an enzyme involved in bothβ-alanine and GABA transamination (Andersen et al., supra).3-Amino-2-methylpropionate transaminase catalyzes the transformationfrom methylmalonate semialdehyde to 3-amino-2-methylpropionate. Thisenzyme has been characterized in Rattus norvegicus and Sus scrofa and isencoded by Abat (Tamaki et al, Methods Enzymol, 324:376-389 (2000)).

GenBank Gene name GI# Accession # Organism SkyPYD4 98626772 ABF58893.1Saccharomyces kluyveri SkUGA1 98626792 ABF58894.1 Saccharomyces kluyveriUGA1 6321456 NP_011533.1 Saccharomyces cerevisiae Abat 122065191P50554.3 Rattus norvegicus Abat 120968 P80147.2 Sus scrofa

2.8.3.a Coenzyme-A transferase.-CoA transferases catalyze reversiblereactions that involve the transfer of a CoA moiety from one molecule toanother. For example, step E of FIG. 10 is catalyzed by a3-oxoadipyl-CoA transferase. In this step, 3-oxoadipate is formed by thetransfer of the CoA group from 3-oxoadipyl-CoA to succinate, acetate, oranother CoA acceptor. Step E of FIG. 11 entails the transfer of a CoAmoiety from another 3-oxoacyl-CoA, 3-oxo-6-aminohexanoyl-CoA. Onecandidate enzyme for these steps is the two-unit enzyme encoded by pcaIand pcaJ in Pseudomonas, which has been shown to have3-oxoadipyl-CoA/succinate transferase activity (Kaschabek et al.,supra). Similar enzymes based on homology exist in Acinetobacter sp.ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)) and Streptomycescoelicolor. Additional exemplary succinyl-CoA:3:oxoacid-CoA transferasesare present in Helicobacter pylori (Corthesy-Theulaz et al., J. Biol.Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al.,Protein. Expr. Purif. 53:396-403 (2007)).

Gene name GI# GeneBank Accession # Organism pcal 24985644 AAN69545.1Pseudomonas putida pcaJ 26990657 NP_746082.1 Pseudomonas putida pcal50084858 YP_046368.1 Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1Acinetobacter sp. ADP1 pcal 21224997 NP_630776.1 Streptomyces coelicolorpcaJ 21224996 NP_630775.1 Streptomyces coelicolor HPAG1_0676 108563101YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB16080949 NP_391777 Bacillus subtilis

A 3-oxoacyl-CoA transferase that can utilize acetate as the CoA acceptoris acetoacetyl-CoA transferase, encoded by the E. coli atoA (alphasubunit) and atoD (beta subunit) genes (Vanderwinkel et al., Biochem.Biophys. Res Commun. 33:902-908 (1968); Korolev et al., ActaCrystallogr. D Biol Crystallogr. 58:2116-2121 (2002)). This enzyme hasalso been shown to transfer the CoA moiety to acetate from a variety ofbranched and linear acyl-CoA substrates, including isobutyrate (Matthieset al., Appl Environ Microbiol 58:1435-1439 (1992)), valerate(Vanderwinkel et al., supra) and butanoate (Vanderwinkel et al., supra).Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncanet al., Appl Environ Microbiol 68:5186-5190 (2002)), Clostridiumacetobutylicum (Cary et al., Appl Environ Microbiol 56:1576-1583(1990)), and Clostridium saccharoperbutylacetonicum (Kosaka et al.,Biosci. Biotechnol Biochem. 71:58-68 (2007)).

Gene name GI# Accession # Organism atoA 2492994 P76459.1 Escherichiacoli K12 atoD 2492990 P76458.1 Escherichia coli K12 actA 62391407YP_226809.1 Corynebacterium glulamicum ATCC 13032 cg0592 62389399YP_224801.1 Corynebacterium glutamicum ATCC 13032 ctfA 15004866NP_149326,1 Clostridium acetobutylicum ctfB 15004867 NP_149327.1Clostridium acetobutylicum ctfA 31075384 AAP42564.1 Clostridiumsaccharoperbutylacetonicum ctfB 31075385 AAP42565.1 Clostridiumsaccharoperbutylacetonicum

The above enzymes may also exhibit the desired activities on adipyl-CoAand adipate (FIG. 10, step K) or 6-aminocaproate and 6-aminocaproyl-CoA(FIG. 10, step Q; FIG. 2, step K). Nevertheless, additional exemplarytransferase candidates are catalyzed by the gene products of cat1, cat2,and cat3 of Clostridium kluyveri which have been shown to exhibitsuccinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferaseactivity, respectively (Seedorf et al., supra; Sohling et al., Eur. J.Biochem. 212:121-127 (1993); Sohling et al., J. Bacteriol. 178:871-880(1996)).

Gene name GI# GeneBank Accession # Organism cat1 729048 P38946.1Clostridium kluyveri cat2 172046066 P38942.2 Ctostridium kluyveri cat3146349050 EDK35586.1 Clostridium ktuyveri

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoAand 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)). Thegenes encoding this enzyme are gctA and gctB. This enzyme has reducedbut detectable activity with other CoA derivatives includingglutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckelet al., Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51(1994)).

Gene name GI# GeneBank Accession # Organism gctA 559392 CAA57199.1Acidaminococcus fermentans gctB 559393 CAA57200.1 Acidaminococcusfermentans

3.1.2.a Thiolester hydrolase (CoA specific). Several eukaryoticacetyl-CoA hydrolases have broad substrate specificity and thusrepresent suitable candidate enzymes for hydrolyzing 3-oxoadipyl-CoA,adipyl-CoA, 3-oxo-6-aminohexanoyl-CoA, or 6-aminocaproyl-CoA (Steps Gand M of FIGS. 10 and 11). For example, the enzyme from Rattusnorvegicus brain (Robinson et al., Biochem. Biophys. Res. Commun.71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA andmalonyl-CoA.

Gene name GI# GenBank Accession # Organism acot12 18543355 NP_570103.1Rattus norvegicus

Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolasewhich has been described to efficiently catalyze the conversion of3-hydroxyisobutyryl-CoA to 3-hydroxyisobutyrate during valinedegradation (Shimomura et al., J Biol Chem. 269:14248-14253 (1994)).Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomuraet al., supra; Shimomura et al., Methods Enzymol. 324:229-240 (2000))and Homo sapiens (Shimomura et al., supra). Candidate genes by sequencehomology include hibch of Saccharomyces cerevisiae and BC 2292 ofBacillus cereus.

Gene name GI# GenBank Accession # Organism hibch 146324906 Q5XIE6.2Rattus norvegicus hibch 146324905 Q6NVY1.2 Homo sapiens hibch 2506374P28817.2 Saccharomyces cerevisiae BC_2292 29895975 AP09256 Bacilluscereus

Yet another candidate hydrolase is the human dicarboxylic acidthioesterase, acot8, which exhibits activity on glutaryl-CoA,adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin etal., J. BioL Chem. 280:38125-38132 (2005)) and the closest E. colihomolog, tesB, which can also hydrolyze a broad range of CoA thiolesters(Naggert et al., J Biol Chem 266:11044-11050 (1991)). A similar enzymehas also been characterized in the rat liver (Deana R., Biochem Int26:767-773 (1992)).

Gene name GI# GenBank Accession # Organism tesB 16128437 NP_414986Escherichia coli acot8 3191970 CAA15502 Homo sapiens acot8 51036669NP_570112 Rattus norvegicus

Other potential E. coli thiolester hydrolases include the gene productsof tesA (Bonner et al., J Biol Chem 247:3123-3133 (1972)), ybgC(Kuznetsova et al., FEMS Microbiol Rev 29:263-279 (2005); Zhuang et al.,FEBS Lett 516:161-163 (2002)), paaI (Song et al., J Biol Chem281:11028-11038 (2006)), and ybdB (Leduc et al., J Bacteriol189:7112-7126 (2007)).

Gene name GI# GenBank Accession # Organism tesA 16128478 NP_415027Escherichia coli ybgC 16128711 NP_415264 Escherichia coli paal 16129357NP_415914 Escherichia coli ybdB 16128580 NP_415129 Escherichia coli

6.3.1.a/6.3.2.a amide synthases/peptide synthases. The direct conversionof 6-aminocaproate to caprolactam (Step S, FIG. 10; Step R, FIG. 11)requires the formation of an intramolecular peptide bond. Ribosomes,which assemble amino acids into proteins during translation, arenature's most abundant peptide bond-forming catalysts. Nonribosomalpeptide synthetases are peptide bond forming catalysts that do notinvolve messenger mRNA (Schwarzer et al., Nat. Prod. Rep. 20:275-287(2003)). Additional enzymes capable of forming peptide bonds includeacyl-CoA synthetase from Pseudomonas chlororaphis (Abe et al., J BiolChem 283:11312-11321 (2008)), gamma-Glutamylputrescine synthetase fromE. coli (Kurihara et al., J Biol Chem 283:19981-19990 (2008)), andbeta-lactam synthetase from Streptomyces clavuligerus (Bachmann et al.,Proc Natl Acad Sci USA 95:9082-9086 (1998); Bachmann et al.,Biochemistry 39:11187-11193 (2000); Miller et al., Nat. Struct. Biol8:684-689 (2001); Miller et al., Proc Natl Acad Sci USA 99:14752-14757(2002); Tahlan et al., Antimicrob. Agents. Chemother. 48:930-939(2004)).

GenBank Gene name GI# Accession # Organism acsA 60650089 BAD90933Pseudomonas chlororaphis puuA 87081870 AAC74379 Escherichia coli bls41016784 Q9R8E3 Streptomyces clavuligerus

4.2.1.a Hydrolyase. Most dehydratases catalyze the α,β-elimination ofwater. This involves activation of the α-hydrogen by anelectron-withdrawing carbonyl, carboxylate, or CoA-thiol ester group andremoval of the hydroxyl group from the β-position. Enzymes exhibitingactivity on substrates with an electron-withdrawing carboxylate groupare excellent candidates for dehydrating 3-hydroxyadipate (FIG. 10, StepI) or 3-hydroxy-6-aminohexanoate (FIG. 11, Step I).

For example, fumarase enzymes naturally catalyze the reversibledehydration of malate to fumarate. E. coli has three fumarases: FumA,FumB, and FumC that are regulated by growth conditions. FumB is oxygensensitive and only active under anaerobic conditions. FumA is activeunder microanaerobic conditions, and FumC is the only active enzyme inaerobic growth (Tseng et al., J Bacteriol 183:461-467 (2001); Woods etal., Biochim Biophys Acta 954:14-26 (1988); Guest et al., J GenMicrobiol 131:2971-2984 (1985)). Additional enzyme candidates are foundin Campylobacter jejuni (Smith et al., Int. J Biochem. Cell Biol31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch.Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi etal., J Biochem. 89:1923-1931 (1981)). Similar enzymes with high sequencehomology include fumI from Arabidopsis thaliana and fumC fromCorynebacterium glutamicum. The MmcBC fumarase from Pelotomaculumthermopropionicum is another class of fumarase with two subunits(Shimoyama et al., FEMS Microbiol Lett 270:207-213 (2007)).

Gene name GI# GeneBank Accession # Organism fumA 81175318 P0AC33Escherichia coli fumB 33112655 P14407 Escherichia coli fumC 120601P05042 Escherichia coli fumC 9789756 O69294 Campylobacter jejuni fumC3062847 BAA25700 Thermus thermophilus fumH 120605 P14408 Rattusnorvegicus fum1 39931311 P93033 Arabidopsis thaliana fumC 39931596Q8NRN8 Corynebacterium glutamicum MmcB 147677691 YP_001211906Pelotomaculum thermopropionicum MmcC 147677692 YP_001211907Pelotomaculum thermopropionicum

Two additional dehydratase candidates are 2-(hydroxymethyl)glutaratedehydratase and dimethylmaleate hydratase, enzymes studied for theirrole in nicontinate catabolism in Eubacterium barkeri (formerlyClostridium barkeri) (Alhapel et al., Proc Natl Acad Sci USA 103:12341-6(2006)). 2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S]-containingenzyme that dehydrates 2-(hydroxymethyl)glutarate to2-methylene-glutarate. This enzyme is encoded by hmd in Eubacteriumbarkeri (Alhapel et al., supra). Similar enzymes with high sequencehomology are found in Bacteroides capillosus, Anaerotruncus colihominis,and Natranaerobius thermophilius. These enzymes are homologous to thealpha and beta subunits of [4Fe-45]-containing bacterial serinedehydratases (e.g., E. coli enzymes encoded by tdcG, sdhB, and sdaA).

Gene name GI# GenBank Accession # Organism hmd 86278275 ABC88407.1Eubacterium barkeri BACCAP_02294 154498305 ZP_02036683.1 Bacteroidescapillosus ANACOL_02527 167771169 ZP_02443222.1 Anaerotruncuscolihominis DSM 17241 NtherDRAFT_2368 169192667 ZP_02852366.1Natranaerobius thermophilus JW/NM-WN-LF

Dimethylmaleate hydratase (EC 4.2.1.85) is a reversible Fe²⁺-dependentand oxygen-sensitive enzyme in the aconitase family that hydratesdimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme isencoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra;Kollmann-Koch et al., Hoppe Seylers. Z. Physiol Chem. 365:847-857(1984)).

Gene name GI# GenBank Accession # Organism dmdA 86278276 ABC88408Eubacterium barkeri dmdB 86278277 ABC88409.1 Eubacterium barkeri

An additional enzyme candidate is 2-methylmalate dehydratase, alsocalled citramalate hydrolyase, a reversible hydrolyase that catalyzesthe alpha, beta elimination of water from citramalate to formmesaconate. This enzyme has been purified and characterized inClostridium tetanomorphum (Wang et al., J. BioL Chem. 244:2516-2526(1969)). The activity of this enzyme has also been detected in severalbacteria in the genera Citrobacter and Morganella in the context of theglutamate degradation VI pathway (Kato et al., Arch. Microbiol168:457-463 (1997)). Genes encoding this enzyme have not been identifiedin any organism to date.

Enzymes exhibiting activity on substrates with an electron-withdrawingCoA-thiol ester group adjacent to the α-hydrogen are excellentcandidates for dehydrating 3-hydroxyadipyl-CoA (FIG. 10, Step C) or3-hydroxy-6-aminohexanoyl-CoA (FIG. 11, Step C). The enoyl-CoAhydratases, phaA and phaB, of P. putida are believed to carry out thehydroxylation of double bonds during phenylacetate catabolism (Oliveraet al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)). The paaA andpaaB from P. fluorescens catalyze analogous transformations (Olivera etal., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)). Lastly, a numberof Escherichia coli genes have been shown to demonstrate enoyl-CoAhydratase functionality including maoC (Park et al., J. Bacteriol.185:5391-5397 (2003)), paaF (Ismail et al., supra; Park et al., Appl.Biochem. Biotechnol 113-116: 335-346 (2004); Park et al., BiotechnolBioeng 86:681-686 (2004)) and paaG (Ismail et al., supra; Park et al.,Appl. Biochem. Biotechnol 113-116:335-346 (2004); Park et al.,Biotechnol Bioeng 86:681-686 (2004)). Crotonase enzymes are additionalcandidates for dehydrating the required 3-hydroxyacyl-CoA moleculesdepicted in FIGS. 10 and 11. These enzymes are required for n-butanolformation in some organisms, particularly Clostridial species, and alsocomprise one step of the 3-hydroxypropionate/4-hydroxybutyrate cycle inthermoacidophilic Archaea of the genera Sulfolobus, Acidianus, andMetallosphaera. Exemplary genes encoding crotonase enzymes can be foundin C. acetobutylicum (Boynton et al., supra), C. kluyveri (Hillmer etal., FEBS Lett. 21:351-354 (1972)), and Metallosphaera sedula (Berg etal., supra) though the sequence of the latter gene is not known.Enoyl-CoA hydratases, which are involved in fatty acid beta-oxidationand/or the metabolism of various amino acids, can also catalyze thehydration of crotonyl-CoA to form 3-hydroxybutyryl-CoA (Roberts et al.,Arch. Microbiol 117:99-108 (1978); Agnihotri et al., Bioorg. Med. Chem.11:9-20 (2003); Conrad et al., J Bacteriol. 118:103-111 (1974)).

Gene name GI# GenBank Accession # Organism paaA 26990002 NP_745427.1Pseudomonas fluorescens paaB 26990001 NP_745426.1 Pseudomonasfluorescens phaA 106636093 ABF82233.1 Pseudomonas putida phaB 106636094ABF82234.1 Pseudomonas putida maoC 16129348 NP_415905.1 Escherichia colipaaF 16129354 NP_415911.1 Escherichia coli paaG 16129355 NP_415912.1Escherichia coli crt 15895969 NP_349318.1 Clostridium acetobutylicumcrt1 153953091 YP_001393856 Clostridium kluyveri DSM 555

6.2.1.a Acid-thiol ligase. Steps F, L, and R of FIG. 10 and Steps F andL of FIG. 11 require acid-thiol ligase or synthetase functionality (theterms ligase, synthetase, and synthase are used herein interchangeablyand refer to the same enzyme class). Exemplary genes encoding enzymeslikely to carry out these transformations include the sucCD genes of E.coli which naturally form a succinyl-CoA synthetase complex. This enzymecomplex naturally catalyzes the formation of succinyl-CoA from succinatewith the concaminant consumption of one ATP, a reaction which isreversible in vivo (Buck et al., Biochem. 24:6245-6252 (1985)). Giventhe structural similarity between succinate and adipate, that is, bothare straight chain dicarboxylic acids, it is reasonable to expect someactivity of the sucCD enzyme on adipyl-CoA.

Gene name GI# GenBank Accession # Organism sucC 16128703 NP_415256.1Escherichia coli sucD 1786949 AAC73823.1 Escherichia coli

Additional exemplary CoA-ligases include the rat dicarboxylate-CoAligase for which the sequence is yet uncharacterized (Vamecq et al.,Biochemical Journal 230:683-693 (1985)), either of the two characterizedphenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al.,Biochem. J. 395:147-155 (2005); Wang et al., Biochem Biophy Res Commun360(2):453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonasputida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)),and the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower etal., J. Bacteriol. 178(14):4122-4130 (1996)). Additional candidateenzymes are acetoacetyl-CoA synthetases from Mus musculus (Hasegawa etal., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens (Ohgamiet al., Biochem Pharmacol 65:989-994 (2003)) which naturally catalyzethe ATP-dependant conversion of acetoacetate into acetoacetyl-CoA.

Gene name GI# GenBank Accession # Organism phl 77019264 CAJ15517.1Penicillium chrysogenum phlB 152002983 ABS19624.1 Penicilliumchrysogenum paaF 22711873 AAC24333.2 Pseudomonas putida bioW 50812281NP_390902.2 Bacillus subtilis AACS 21313520 NP_084486.1 Mus musculusAACS 31982927 NP_0764172 Homo sapiens

ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is anothercandidate enzyme that couples the conversion of acyl-CoA esters to theircorresponding acids with the concurrent synthesis of ATP. Severalenzymes with broad substrate specificities have been described in theliterature. ACD I from Archaeoglobus fulgidus, encoded by AF1211, wasshown to operate on a variety of linear and branched-chain substratesincluding acetyl-CoA, propionyl-CoA, butyryl-CoA, acetate, propionate,butyrate, isobutyryate, isovalerate, succinate, fumarate, phenylacetate,indoleacetate (Musfeldt et al., J Bacteriol 184:636-644 (2002)). Theenzyme from Haloarcula marismortui (annotated as a succinyl-CoAsynthetase) accepts propionate, butyrate, and branched-chain acids(isovalerate and isobutyrate) as substrates, and was shown to operate inthe forward and reverse directions (Brasen et al., Arch Microbiol182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophiliccrenarchaeon Pyrobaculum aerophilum showed the broadest substrate rangeof all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA(preferred substrate) and phenylacetyl-CoA (Brasen et al., supra). Theenzymes from A. fulgidus, H. marismortui and P. aerophilum have all beencloned, functionally expressed, and characterized in E. coli (Musfeldtet al., supra; Brasen et al., supra).

Gene name GI# GenBank Accession # Organism AF1211 11498810 NP_070039.1Archaeoglobus fulgidus DSM 4304 scs 55377722 YP_135572.1 Haloarculamarismortui ATCC 43049 PAE3250 18313937 NP_560604.1 Pyrobaculumaerophilum str. IM2

Yet another option is to employ a set of enzymes with net ligase orsynthetase activity. For example, phosphotransadipylase and adipatekinase enzymes are catalyzed by the gene products of buk1, buk2, and ptbfrom C. acetobutylicum (Walter et al., Gene 134:107-111 (1993); Huang etal., J. Mol. Microbiol. Biotechnol. 2:33-38 (2000)). The ptb geneencodes an enzyme that can convert butyryl-CoA into butyryl-phosphate,which is then converted to butyrate via either of the buk gene productswith the concomitant generation of ATP.

GenBank Gene name GI# Accession # Organism ptb 15896327 NP_349676Clostridium acetobutylicum buk1 15896326 NP_349675 Clostridiumacetobutylicum buk2 20137415 Q97II1 Clostridium acetobutylicum

No enzyme required—Spontaneous cyclization. 6-Aminocaproyl-CoA willcyclize spontaneously to caprolactam, thus eliminating the need for adedicated enzyme for this step. A similar spontaneous cyclization isobserved with 4-aminobutyryl-CoA which forms pyrrolidinone (Ohsugi etal., J Biol Chem 256:7642-7651 (1981)).

Microbiol organisms may also be generated that are capable of producinghexamethylenediamine from acetyl-CoA and succinyl-CoA and as shown inFIG. 10 that starts from acetyl-CoA and succinyl-CoA. E. coli provides agood host for generating a non-naturally occurring microorganism capableof producing hexamethylenediamine. While E. coli may be used to describethis pathway, it should be understood that any microorganism can beadapted to generate such pathway. To generate an E. coli strainengineered to produce hexamethylenediamine, nucleic acids encoding therequisite enzymes are expressed in E. coli using well known molecularbiology techniques (see, for example, Sambrook, supra, 2001; Ausubel,supra, 1999). In particular, the paaJ (NP-415915.1), paaH (NP-415913.1),and maoC (NP-415905.1) genes encoding the 3-oxoadipyl-CoA thiolase,3-oxoadipyl-CoA reductase, and 3-hydroxyadipyl-CoA dehydrataseactivities, respectively, are cloned into the pZE13 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd(NP-349317.1) and eftAB (NP-349315.1 and NP-349316.1) genes encoding5-carboxy-2-pentenoyl-CoA reductase activity are cloned into the pZA33vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter.Lastly, the acrl (YP-047869.1), gabT (NP-417148.1), bioW (NP-390902.2),and ygjG (NP-417544) genes encoding adipyl-CoA reductase (aldehydeforming), 6-aminocaproyl-CoA reductase (aldehyde forming),6-aminocaproic acid transaminase, 6-aminocaproyl-CoA synthase, andhexamethylenediamine transaminase activities are cloned into a thirdcompatible plasmid, pZS23, under the PA1/lacO promoter. pZS23 isobtained by replacing the ampicillin resistance module of the pZS13vector (Expressys, Ruelzheim, Germany) with a kanamycin resistancemodule by well-known molecular biology techniques. The three sets ofplasmids are transformed into E. coli strain MG1655 to express theproteins and enzymes required for hexamethylenediamine synthesis.

In other examples, hexamethylenediamine can be produced via a pathwayfor converting acetyl-CoA and 4-aminobutyryl-CoA to 6-Aminocaproyl-CoA

Another pathway to produce hexamethylenediamine is from acetyl-CoA and4-aminobutyryl-CoA.

The paaJ (NP-415915.1), paaH (NP-415913.1), and maoC (NP-415905.1) genesencoding the 3-oxo-6-aminohexanoyl-CoA thiolase,3-oxo-6-aminohexanoyl-CoA reductase, 3-hydroxy-6-aminohexanoyl-CoAdehydratase activities, respectively, can be cloned into the pZE13vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Inaddition, the bcd (NP-349317.1), etfAB (NP-349315.1 and NP-349316.1),acrl (YP-047869.1), and ygjG (NP-417544) genes encoding6-aminohex-2-enoyl-CoA reductase, 6-aminocaproyl-CoA reductase (aldehydeforming), and hexamethylenediamine transaminase activities are clonedinto the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. Lastly, the sucD (NP-904963.1), gabT (NP-417148.1), and cat2(P38942.2) genes encoding succinyl-CoA reductase (aldehyde forming),GABA transaminase, and 4-aminobutyryl-CoA/acyl-CoA transferaseactivities are cloned into a third compatible plasmid, pZS23, under thePA1/lacO promoter, to increase the availability of 4-aminobutyryl-CoA.pZS23 is obtained by replacing the ampicillin resistance module of thepZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin resistancemodule by well-known molecular biology techniques. The three sets ofplasmids are transformed into E. coli strain MG1655 to express theproteins and enzymes required for hexamethylenediamine synthesis.

Hexamethylenediamine may also be produced from 6-Aminocaproate (6-ACA).This pathway involves activation of the acid group by phosphorylationand/or acylation. Acetylation of the terminal amino group providesprotection from spontaneous cyclization of pathway intermediates.

Several pathways for producing HMD from 6-aminocaproate are detailed inFIG. 13. All routes entail activation of the carboxylic acid group,followed by reduction and transamination. In three routes,6-aminocaproate is activated directly while in other routes, theterminal amine group is protected by N-acetylation to preventspontaneous cyclization.

In one route, 6-aminocaproate is phosphorylated to 6-AHOP by6-aminocaproate kinase (FIG. 13, Step A). 6-AHOP is then reduced to6-aminocaproic semialdehyde (FIG. 13, Step B) and subsequentlytransaminated (FIG. 13, Step C) by an aminotransferase or an aminatingoxidoreductase.

Alternately, 6-AHOP is converted to 6-aminocaproyl-CoA by anacyltransferase (FIG. 13, Step L). 6-Aminocaproyl-CoA is then reduced to6-aminocaproic semialdehyde by a CoA-dependent aldehyde dehydrogenase(FIG. 13, Step N). HMD is then formed by transamination of6-aminocaproic semialdehyde by an aminotransferase or aminatingoxidoreductase (FIG. 13, Step C).

In yet another route, 6-aminocaproate is first activated to a CoAderivative by a CoA transferase or CoA ligase (FIG. 13, Step M). Theproduct, 6-aminocaproyl-CoA, may spontaneously cyclize, or be convertedto 6-aminocaproic semialdehyde by an aldehyde-forming CoA-dependentaldehyde dehydrogenase (FIG. 13, Step N). 6-Aminocaproic semialdehyde isconverted to HMD by an aminotransferase or an aminating oxidoreductase(FIG. 13, Step C).

Additional routes proceed from 6-acetamidohexanoate, the acetylatedproduct of 6-aminocaproate N-acetyltransferase. 6-Acetamidohexanoate isconverted to 6-acetamidohexanal by different routes (described below).In the final two steps of these routes, 6-acetamidohexanal is firstconverted to 6-acetamidohexanamine by an aminotransferase or anaminating oxidoreductase (FIG. 13, Step G). 6-Acetamidohexanamine issubsequently converted to HMD by an amide hydrolase or anN-acetyltransferase (FIG. 13, Step H).

In one route, 6-acetamidohexanoate is phosphorylated by6-acetamidohexanoate kinase (FIG. 13, Step E). The product, 6-AAHOP, isreduced to form 6-acetamidohexanal (FIG. 13, Step F), which is thenconverted to HMD as described above.

In another route, 6-acetamidohexanoate is activated to6-acetamidohexanoyl-CoA by a CoA transferase or CoA ligase (FIG. 13,Step I). The CoA derivative is then reduced to 6-acetamidohexanal by analdehyde-forming CoA-dependent oxidoreductase (FIG. 13, Step J).6-acetamidohexanal is then converted to HMD as described above.

Alternately, 6-acetamidohexanoate is phosphorylated to 6-AAHOP (FIG. 13,Step E) and subsequently converted to 6-acetamidohexanoyl-CoA by anacyltransferase (FIG. 13, Step K). 6-Acetamidohexanoyl-CoA is thenreduced to HMD as described previously.

The transformations depicted in FIGS. 12 and 13 fall into the generalcategories of transformations shown in Table 9. Below is described anumber of biochemically characterized genes in each category.Specifically listed are genes that can be applied to catalyze theappropriate transformations in FIGS. 12-13 when properly cloned andexpressed.

Table 9 shows the enzyme types useful to convert common centralmetabolic intermediates into 6-aminocaproate and hexamethylenediamine.The first three digits of each label correspond to the first threeEnzyme Commission number digits which denote the general type oftransformation independent of substrate specificity.

TABLE 9 LABEL FUNCTION 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde)1.2.1.c Oxidoreductase (2-ketoacid to acyl-CoA) 1.2.1.d Oxidoreductase(phosphonic acid to aldehyde) 1.3.1.a Oxidoreductase (alkene to alkane)1.4.1.a Oxidoreductase (ketone or aldehyde to amino) 2.3.1.aAcyltransferase (transferring CoA to phospho) 2.3.1.c Acyltransferase(N-acetyltransferase) 2.3.1.d Acyltransferase (formateC-acyltransferase) 2.6.1.a Aminotransferase 2.7.2.a Phosphotransferase(carboxy acceptor) 2.8.3.a Coenzyme-A transferase 3.5.1.a Hydrolase(acting on linear amides) 4.1.1.a Carboxy-lyase 4.1.2.a Aldehyde-lyase4.2.1.a Hydro-lyase 6.2.1.a Acid-thiol ligase

1.2.1.b Oxidoreductase (acyl-CoA to aldehyde). The transformations of6-acetamidohexanoyl-CoA to 6-acetamidohexanal (FIG. 13, Step J) and6-aminocaproyl-CoA to 6-aminocaproic semialdehyde (FIG. 13, Step N) arecatalyzed by CoA-dependent oxidoreductase enzyme in the EC class 1.2.1.Adipyl-CoA is converted to adipate semialdehyde by adipyl-CoAoxidoreductase, an enzyme with similar functionality (FIG. 12, Step O).Succinic semialdehyde dehydrogenase, an enzyme that forms FIG. 12precursor succinic semialdehyde from succinyl-CoA, is also aCoA-dependent oxidoreductase. Oxidoreductases in the EC class 1.2.1.—arecapable of reducing an acyl-CoA to its corresponding aldehyde. Exemplarygenes that encode such enzymes include the Acinetobacter calcoaceticusacrl encoding a fatty acyl-CoA reductase (Reiser and Somerville, Journalof Bacteriology 179:2969-2975 (1997)), the Acinetobacter sp. M-1 fattyacyl-CoA reductase (Ishige et al., Appl. Environ. Microbiol.68:1192-1195 (2002)), and a CoA- and NADP-dependent succinicsemialdehyde dehydrogenase encoded by the sucD gene in Clostridiumkluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)).SucD of P. gingivalis is another succinic semialdehyde dehydrogenase(Takahashi et al., J. Bacteriol. 182:4704-4710 (2000)). The acylatingacetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yetanother candidate as it has been demonstrated to oxidize and acylateacetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde andformaldehyde (Powlowski et al., J. Bacteriol. 175:377-385 (1993)). Inaddition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhEin Leuconostoc mesenteroides has been shown to oxidize the branchedchain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., J.Gen. Appl. Microbiol. 18:43-55 (1972); and Koo et al., Biotechnol Lett.27:505-510 (2005)).

GenBank Gene name GI# Accession # Organism acr1 50086359 YP_047869.1Acinetobacter calcoaceticus acr1 1684886 AAC45217 Acinetobacter baylyiacr1 18857901 BAB85476.1 Acinetobacter sp. Strain M-1 sucD 172046062P38947.1 Clostridium kluyveri sucD 34540484 NP_904963.1 Porphyromonasgingivalis bphG 425213 BAA03892.1 Pseudomonas sp adhE 55818563AAV66076.1 Leuconostoc mesenteroides

An additional enzyme that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archaeal bacteria (Berg et al., Science 318:1782-1786(2007); and Thauer, R. K., Science. 318:1732-1733 (2007)). The enzymeutilizes NADPH as a cofactor and has been characterized inMetallosphaera and Sulfolobus sp (Alber et al., J. Bacteriol.188:8551-8559 (2006); and Hugler et al., J. Bacteriol. 184:2404-2410(2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula(Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg et al.,Science. 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductasefrom Sulfolobus tokodaii was cloned and heterologously expressed in E.coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)). This enzyme hasalso been shown to catalyze the conversion of methylmalonyl-CoA to itscorresponding aldehyde (WIPO Patent Application WO/2007/141208 KindCode: A2). Although the aldehyde dehydrogenase functionality of theseenzymes is similar to the bifunctional dehydrogenase from Chloroflexusaurantiacus, there is little sequence similarity. Both malonyl-CoAreductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional gene candidates can be found by sequencehomology to proteins in other organisms including Sulfolobussolfataricus and Sulfolobus acidocaldarius and have been listed below.Yet another candidate for CoA-acylating aldehyde dehydrogenase is theaid gene from Clostridium beijerinckii (Toth et al., Appl EnvironMicrobiol 65:4973-4980 (1999)). This enzyme has been reported to reduceacetyl-CoA and butyryl-CoA to their corresponding aldehydes. This geneis very similar to eutE that encodes acetaldehyde dehydrogenase ofSalmonella typhimurium and E. coli (Toth et al., Appl Environ Microbiol65:4973-4980 (1999)).

Gene name GI# GenBank Accession # Organism Msed_0709 146303492YP_001190808.1 Metallosphaera sedula mcr 15922498 NP_378167.1 Sulfolobustokodaii asd-2 15898958 NP_343563.1 Sulfolobus solfataricus Saci_237070608071 YP_256941.1 Sulfolobus acidocaldarius Ald 49473535 AAT66436Clostridium beijerinckii eutE 687645 AAA80209 Salmonella typhimuriumeutE 2498347 P77445 Escherichia coli

1.2.1.c Oxidoreductase (2-ketoacid to acyl-CoA). Several transformationsin FIG. 12 require conversion of a 2-ketoacid to an acyl-CoA (Steps L, Pand Q) by an enzyme in the EC class 1.2.1. Such reactions are catalyzedby multi-enzyme complexes that catalyze a series of partial reactionswhich result in acylating oxidative decarboxylation of 2-keto-acids.Exemplary enzymes include 1) branched-chain 2-keto-acid dehydrogenase,2) alpha-ketoglutarate dehydrogenase, and 3) the pyruvate dehydrogenasemultienzyme complex (PDHC). Each of the 2-keto-acid dehydrogenasecomplexes occupies key positions in intermediary metabolism, and enzymeactivity is typically tightly regulated (Fries et al., Biochemistry42:6996-7002 (2003)). The enzymes share a complex but common structurecomposed of multiple copies of three catalytic components:alpha-ketoacid decarboxylase (E1), dihydrolipoamide acyltransferase (E2)and dihydrolipoamide dehydrogenase (E3). The E3 component is sharedamong all 2-keto-acid dehydrogenase complexes in an organism, while theE1 and E2 components are encoded by different genes. The enzymecomponents are present in numerous copies in the complex and utilizemultiple cofactors to catalyze a directed sequence of reactions viasubstrate channeling. The overall size of these dehydrogenase complexesis very large, with molecular masses between 4 and 10 million Da (i.e.larger than a ribosome).

Activity of enzymes in the 2-keto-acid dehydrogenase family is normallylow or limited under anaerobic conditions in E. coli. Increasedproduction of NADH (or NADPH) could lead to a redox-imbalance, and NADHitself serves as an inhibitor to enzyme function. Engineering effortshave increased the anaerobic activity of the E. coli pyruvatedehydrogenase complex (Kim et al., Appl. Environ. Microbiol.73:1766-1771 (2007); Kim et al., J. Bacteriol. 190:3851-3858 (2008); andZhou et al., Biotechnol. Lett. 30:335-342 (2008)). For example, theinhibitory effect of NADH can be overcome by engineering an H322Ymutation in the E3 component (Kim et al., J. Bacteriol. 190:3851-3858(2008)). Structural studies of individual components and how they worktogether in complex provide insight into the catalytic mechanisms andarchitecture of enzymes in this family (Aevarsson et al., Nat. Struct.Biol. 6:785-792 (1999); and Zhou et al., Proc. Natl. Acad. Sci. U.S. A98:14802-14807 (2001)). The substrate specificity of the dehydrogenasecomplexes varies in different organisms, but generally branched-chainketo-acid dehydrogenases have the broadest substrate range.

Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate tosuccinyl-CoA and is the primary site of control of metabolic fluxthrough the TCA cycle (Hansford, Curr. Top. Bioenerg. 10:217-278(1980)). Encoded by genes sucA, sucB and Ipd in E. coli, AKGD geneexpression is downregulated under anaerobic conditions and during growthon glucose (Park et al., Mol. Microbiol. 15:473-482 (1995)). Althoughthe substrate range of AKGD is narrow, structural studies of thecatalytic core of the E2 component pinpoint specific residuesresponsible for substrate specificity (Knapp et al., J. Mol. Biol.280:655-668 (1998)). The Bacillus subtilis AKGD, encoded by odhAB (E1and E2) and pdhD (E3, shared domain), is regulated at thetranscriptional level and is dependent on the carbon source and growthphase of the organism (Resnekov et al., Mol. Gen. Genet. 234:285-296(1992)). In yeast, the LPD1 gene encoding the E3 component is regulatedat the transcriptional level by glucose (Roy and Dawes, J. Gen.Microbiol. 133:925-933 (1987)). The E1 component, encoded by KGD1, isalso regulated by glucose and activated by the products of HAP2 and HAP3(Repetto and Tzagoloff, Mol. Cell. Biol. 9:2695-2705 (1989)). The AKGDenzyme complex, inhibited by products NADH and succinyl-CoA, iswell-studied in mammalian systems, as impaired function of has beenlinked to several neurological diseases (Tretter and dam-Vizi, Philos.Trans. R. Soc. Lond B Biol. Sci. 360:2335-2345 (2005)).

Gene name GI# GenBank Accession # Organism sucA 16128701 NP_415254.1Escherichia coli sucB 16128702 NP_415255.1 Escherichia coli lpd 16128109NP_414658.1 Escherichia coli odhA 51704265 P23129.2 Bacillus subtilisodhB 129041 P16263.1 Bacillus subtilis pdhD 118672 P21880.1 Bacillussubtilis KGD1 6322066 NP_012141.1 Saccharomyces cerevisiae KGD2 6320352NP_010432.1 Saccharomyces cerevisiae LPD1 14318501 NP_116635.1Saccharomyces cerevisiae

Branched-chain 2-keto-acid dehydrogenase complex (BCKAD), also known as2-oxoisovalerate dehydrogenase, participates in branched-chain aminoacid degradation pathways, converting 2-keto acids derivatives ofvaline, leucine and isoleucine to their acyl-CoA derivatives and CO₂.The complex has been studied in many organisms including Bacillussubtilis (Wang et al., Eur. J. Biochem. 213:1091-1099 (1993)), Rattusnorvegicus (Namba et al., J. Biol. Chem. 244:4437-4447 (1969)) andPseudomonas putida (Sokatch et al., J. Bacteriol. 148:647-652 (1981)).In Bacillus subtilis the enzyme is encoded by genes pdhD (E3 component),bfmBB (E2 component), bfmBAA and bfmBAB (E1 component) (Wang et al.,Eur. J. Biochem. 213:1091-1099 (1993)). In mammals, the complex isregulated by phosphorylation by specific phosphatases and proteinkinases. The complex has been studied in rat hepatocites (Chicco et al.,J. Biol. Chem. 269:19427-19434 (1994)) and is encoded by genes Bckdha(E1 alpha), Bckdhb (E1 beta), Dbt (E2), and Dld (E3). The E1 and E3components of the Pseudomonas putida BCKAD complex have beencrystallized (Aevarsson et al., Nat. Struct. Biol. 6:785-792 (1999); andMattevi et al., Science. 255:1544-1550 (1992)) and the enzyme complexhas been studied (Sokatch et al., J. Bacteriol. 148:647-652 (1981)).Transcription of the P. putida BCKAD genes is activated by the geneproduct of bkdR (Hesslinger et al., Mol. Microbiol. 27:477-492 (1998)).In some organisms including Rattus l norvegicus (Paxton et al., Biochem.J. 234:295-303 (1986)) and Saccharomyces cerevisiae (Sinclair et al.,Biochem. Mol. Biol. int. 31: 911-922 (1993)), this complex has beenshown to have a broad substrate range that includes linear oxo-acidssuch as 2-oxobutanoate and alpha-ketoglutarate, in addition to thebranched-chain amino acid precursors. The active site of the bovineBCKAD was engineered to favor alternate substrate acetyl-CoA (Meng andChuang, Biochemistry. 33:12879-12885 (1994)).

Gene name GI# GenBank Accession # Organism bfmBB 16079459 NP_390283.1Bacillus subtilis bfmBAA 16079461 NP_390285.1 Bacillus subtilis bfmBAB16079460 NP_390284.1 Bacillus subtilis pdhD 118672 P21880.1 Bacillussubtilis lpdV 118677 P09063.1 Pseudomonas putida bkdB 129044 P09062.1Pseudomonas putida bkdA1 26991090 NP_746515.1 Pseudomonas putida bkdA226991091 NP_746516.1 Pseudomonas putida Bckdha 77736548 NP_036914.1Rattus norvegicus Bckdhb 158749538 NP_062140.1 Rattus norvegicus Dbt158749632 NP_445764.1 Rattus norvegicus Dld 40786469 NP_955417.1 Rattusnorvegicus

The pyruvate dehydrogenase complex, catalyzing the conversion ofpyruvate to acetyl-CoA, has also been extensively studied. In the E.coli enzyme, specific residues in the E1 component are responsible forsubstrate specificity (Bisswanger, J Biol Chem. 256:815-822 (1981);Bremer, Eur. J Biochem. 8:535-540 (1969); and Gong et al., J Biol Chem.275:13645-13653 (2000)). As mentioned previously, enzyme engineeringefforts have improved the E. coli PDH enzyme activity under anaerobicconditions (Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007);Kim et al., J. Bacteriol. 190:3851-3858 (2008)); and Zhou et al.,Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coli PDH,the B. subtilis complex is active and required for growth underanaerobic conditions (Nakano et al., J. Bacteriol. 179:6749-6755(1997)). The Klebsiella pneumoniae PDH, characterized during growth onglycerol, is also active under anaerobic conditions (Menzel et al., J.Biotechnol. 56:135-142 (1997)). Crystal structures of the enzyme complexfrom bovine kidney (Zhou et al., Proc. Natl. Acad. Sci. U.S. A98:14802-14807 (2001)) and the E2 catalytic domain from Azotobactervinelandii are available (Mattevi et al., Science. 255:1544-1550(1992)). Some mammalian PDH enzymes complexes can react on alternatesubstrates such as 2-oxobutanoate, although comparative kinetics ofRattus norvegicus PDH and BCKAD indicate that BCKAD has higher activityon 2-oxobutanoate as a substrate (Paxton et al., Biochem. J. 234:295-303(19861.

Gene name GI# GenBank Accession # Organism aceE 16128107 NP_414656.1Escherichia coli aceF 16128108 NP_414657.1 Escherichia coli lpd 16128109NP_414658.1 Escherichia coli pdhA 3123238 P21881.1 Bacillus subtilispdhB 129068 P21882.1 Bacillus subtilis pdhC 129054 P21883.2 Bacillussubtilis pdhD 118672 P21880.1 Bacillus subtilis ace 152968699YP_001333808.1 Klebsiella pneumonia aceF 152968700 YP_001333809.1Klebsiella pneumonia lpdA 152968701 YP_001333810.1 Klebsiella pneumoniaPdha1 124430510 NP_001004072.2 Rattus norvegicus Pdha2 16758900NP_446446.1 Rattus norvegicus Dlat 78365255 NP_112287.1 Rattusnorvegicus Dld 40786469 NP_955417.1 Rattus norvegicus

As an alternative to the large multienzyme 2-keto-acid dehydrogenasecomplexes described above, some anaerobic organisms utilize enzymes inthe 2-ketoacid oxidoreductase family (OFOR) to catalyze acylatingoxidative decarboxylation of 2-keto-acids. Unlike the dehydrogenasecomplexes, these enzymes contain iron-sulfur clusters, utilize differentcofactors, and use ferredoxin or flavodoxin as electron acceptors inlieu of NAD(P)H. While most enzymes in this family are specific topyruvate as a substrate (POR) some 2-keto-acid:ferredoxinoxidoreductases have been shown to accept a broad range of 2-ketoacidsas substrates including alpha-ketoglutarate and 2-oxobutanoate (Fukudaand Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002); and Zhang et al.,J. Biochem. 120:587-599 (1996)). One such enzyme is the OFOR from thethermoacidophilic archaeon Sulfolobus tokodaii 7, which contains analpha and beta subunit encoded by gene ST2300 (Fukuda and Wakagi,Biochim. Biophys. Acta 1597:74-80 (2002); and Zhang et al., J. Biochem.120:587-599 (1996)). A plasmid-based expression system has beendeveloped for efficiently expressing this protein in E. coli (Fukuda etal., Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved insubstrate specificity were determined (Fukuda and Wakagi, Biochim.Biophys. Acta 1597:74-80 (2002)). Two OFORs from Aeropyrum pernix str.K1 have also been recently cloned into E. coli, characterized, and foundto react with a broad range of 2-oxoacids (Nishizawa et al., FEBS Lett.579:2319-2322 (2005)). The gene sequences of these OFOR candidates areavailable, although they do not have GenBank identifiers assigned todate. There is bioinformatic evidence that similar enzymes are presentin all archaea, some anaerobic bacteria and amitochondrial eukarya(Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). Thisclass of enzyme is also interesting from an energetic standpoint, asreduced ferredoxin could be used to generate NADH by ferredoxin-NADreductase (Petitdemange et al., Biochim. Biophys. Acta 421:334-337(1976)). Also, since most of the enzymes are designed to operate underanaerobic conditions, less enzyme engineering may be required relativeto enzymes in the 2-keto-acid dehydrogenase complex family for activityin an anaerobic environment.

Gene name GI# GenBank Accession # Organism ST2300 15922633 NP_378302.1Sulfolobus tokodaii 7

1.2.1.d Oxidoreductase (phosphonic acid to aldehyde). The reduction of aphosphonic acid to its corresponding aldehyde is catalyzed by anoxidoreductase in the EC class 1.2.1. Steps B and F in FIG. 13 requiresuch an enzyme for the reduction of 6-AHOP and 6-AAHOP to theircorresponding aldehydes. These reactions are not catalyzed by knownenzymes, but a similar reaction is catalyzed by aspartate semialdehydedehydrogenase (ASD, EC 1.2.1.11): the NADPH-dependent reduction of4-aspartyl phosphate to aspartate-4-semialdehyde. ASD participates inamino acid biosynthesis and recently has been studied as anantimicrobial target (Hadfield et al., Biochemistry 40:14475-14483(2001)). The E. coli ASD structure has been solved (Hadfield et al., J.Mol. Biol. 289:991-1002 (1999)) and the enzyme has been shown to acceptthe alternate substrate beta-3-methylaspartyl phosphate (Shames et al.,J. Biol. Chem. 259:15331-15339 (1984)). The Haemophilus influenzaeenzyme has been the subject of enzyme engineering studies to altersubstrate binding affinities at the active site (Blanco et al., ActaCrystallogr. D. Biol. Crystallogr. 60:1388-1395 (2004); and Blanco etal., Acta Crystallogr. D. Biol. Crystallogr. 60:1808-1815 (2004)). OtherASD candidates are found in Mycobacterium tuberculosis (Shafiani et al.,J Appl Microbiol 98:832-838 (2005)), Methanococcus jannaschii (Faehnleet al., J Mol. Biol. 353:1055-1068 (2005)), and the infectiousmicroorganisms Vibrio cholera and Heliobacter pylori (Moore et al.,Protein Expr. Purif. 25:189-194 (2002)). A related enzyme candidate isacetylglutamylphosphate reductase (EC 1.2.1.38), an enzyme thatnaturally reduces acetylglutamylphosphate toacetylglutamate-5-semialdehyde, found in S. cerevisiae (Pauwels et al.,Eur. J Biochem. 270:1014-1024 (2003)), B. subtilis (O'Reilly and Devine,Microbiology 140 (Pt 5):1023-1025 (1994)) and other organisms.

Gene name GI# GenBank Accession # Organism Asd 16131307 NP_417891.1Escherichia coli Asd 68249223 YP_248335.1 Haemophilus influenzae Asd1899206 AAB49996 Mycobacterium tuberculosis VC2036 15642038 NP_231670Vibrio cholera Asd 210135348 YP_002301787.1 Heliobacter pylori ARG5,66320913 NP_010992.1 Saccharomyces cerevisiae argC 16078184 NP_389001.1Bacillus subtilis

1.3.1.a Oxidoreductase (alkene to alkane). Several transformations fallinto the category of oxidoreductases that reduce an alkene to an alkane(EC 1.3.1.-). For example, Steps C, G, K and N in FIG. 12, catalyzed byOHED reductase, 6-OHE reductase, 2-AHE reductase and2,3-dehydroadipyl-CoA reductase, respectively, fall into this category.Enone reductase, alkenal reductase, and enoate reductase enzymes aresuitable enzyme candidates for catalyzing the transformations of StepsC, G and K. Enoyl-CoA reductase enzymes catalyze the conversion of2,3-dehydroadipyl-CoA to adipyl-CoA (Step N).

Enzymes with enone reductase activity have been identified inprokaryotes, eukaryotes and plants (Shimoda et al., Bulletin of thechemical Society of Japan 77:2269-2 (2004); and Wanner and Tressl, Eur.J Biochem. 255:271-278 (1998)). Two enone reductases from the cytosolicfraction of Saccharomyces cerevisiae were purified and characterized,and found to accept a variety of alkenals (similar to 6-OHE) and enoylketones (similar to OHED) as substrates (Wanner and Tressl, Eur. JBiochem. 255:271-278 (1998)). Genes encoding these enzymes have not beenidentified to date. Cell extracts of cyanobacterium Synechococcus sp.PCC7942 reduced a variety enone substrates to their corresponding alkylketones (Shimoda et al., Bulletin of the chemical Society of Japan77:2269-2 (2004)). Genes have not been associated with this activity inthis organism. Enone reductases in other organisms can also catalyzethis transformation.

A recombinant NADPH-dependent enone reductase from Nicotiana tabacum,encoded by NtRed1, was functionally expressed and characterized in E.coli (Matsushima et al., Bioorganic Chemistry 36:23-28 (2008)). Thisreductase was functional on the exocyclic enoyl ketone pulegone(Matsushima et al., Bioorganic Chemistry 36:23-28 (2008)). An enzymecandidate in S. cerevisiae at the locus YML131W, bears 30% identity toNtRed1(evalue=1e-26). The amino acid sequence of NtRed1 sharessignificant homology with 2-alkenal reductase from Arabidopsis thaliana,zeta-crystallin homolog from A. thaliana, pulegone reductase from Menthepiperita and phenylpropenal alkene reductase from Pinus taeda. Theseenzymes are known to catalyze the reduction of alkenes ofα,β-unsaturated ketones and aldehydes.

Gene name GI# GenBank Accession # Organism NtRed1 6692816 BAA89423Nicotiana tabacum YML131W 45269874 AAS56318.1 Saccharomyces cerevisiaeAtDBR1 15237888 NP-197199 Arabidopsis thaliana P2 886430 CAA89262Arabidopsis thaliana PulR 34559418 AAQ75423 Menthe piperita PtPPDBR110816011 ABG91753 Pinus taeda

2-Alkenal reductase catalyzes the reduction of α,β-unsaturated doublebonds of aldehydes and ketones. A barley alkenal hydrogenase ALH1 wasidentified with activity for a range of α,β-unsaturated ketones andaldehydes including trans-2-nonenal, 2-hexenal, traumatin and1-octene-3-one (Hambraeus and Nyberg, J Agric. Food Chem. 53:8714-8721(2005)). The Hordeum vulgare ALH1 cDNA was cloned expressed in E. coli(Hambraeus and Nyberg, J Agric. Food Chem. 53:8714-8721 (2005)).

Gene name GI# GenBank Accession # Organism ALH1 62765876 AAX99161Hordeum vulgare ALH1 195652571 ACG45753 Zea mays

2-Enoate reductase enzymes are known to catalyze the NAD(P)H-dependentreduction of a wide variety of α,β-unsaturated carboxylic acids andaldehydes (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). In therecently published genome sequence of C. kluyveri, 9 coding sequencesfor enoate reductases were reported, out of which one has beencharacterized (Seedorf et al., Proc. Natl. Acad. Sci U.S. A105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and M.thermoaceticum have been cloned and sequenced and show 59% identity toeach other. The former gene is also found to have approximately 75%similarity to the characterized gene in C. kluyveri (Giesel and Simon,Arch. Microbiol 135:51-57 (1983)). It has been reported based on thesesequence results that enr is very similar to the dienoyl CoA reductasein E coil (fadH) (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)).The C. thermoaceticum enr gene has also been expressed in acatalytically active form in E. coli (Rohdich et al., J. Biol. Chem.276:5779-5787 (2001)).

Gene name GI# GenBank Accession # Organism Enr 169405742 ACA54153.1Clostridium botulinum A3 str Enr 2765041 CAA71086.1 Clostridiumtyrobutyricum Enr 3402834 CAA76083.1 Clostridium kluyveri Enr 83590886YP_430895.1 Moorella thermoacetica fadH 16130976 NP_417552.1 Escherichiacoli

Another candidate enoate reductase is 3-oxoadipate oxidoreductase(maleylacetate reductase), an enzyme catalyzing the reduction of2-maleylacetate (4-oxohex-2-enedioate) to 3-oxoadipate. The enzymeactivity was identified and characterized in Pseudomonas sp. strain B13(Kaschabek and Reineke, J. Bacteriol. 177:320-325 (1995); and Kaschabek.and Reineke, J. Bacteriol. 175:6075-6081 (1993)), and the coding genewas cloned and sequenced (Kasberg et al., J. Bacteriol. 179:3801-3803(1997)). Candidate genes for 3-oxoadipate oxidoreductase include cicEgene from Pseudomonas sp. strain B13 (Kasberg et al., J. Bacteriol.179:3801-3803 (1997)), macA gene from Rhodococcus opacus (Seibert etal., J. Bacteriol. 180:3503-3508 (1998)), and macA gene from Ralstoniaeutropha (also known as Cupriavidus necator) (Seibert et al.,Microbiology 150:463-472 (2004)).

Gene name GI# GenBank Accession # Organism clcE 3913241 O30847.1Pseudomonas sp. strain B13 macA 7387876 O84992.1 Rhodococcus opacus macA5916089 AAD55886 Cupriavidus necator

Enoyl-CoA reductase enzymes are suitable enzymes for catalyzing thereduction of 2,3-dehydroadipyl-CoA to adipyl-CoA (FIG. 12, Step N). Oneexemplary enoyl-CoA reductase is the gene product of bcd from C.acetobutylicum (Atsumi et al., Metab Eng 10:305-311 (2008); and Boyntonet al., J. Bacteriol. 178:3015-3024 (1996)), which naturally catalyzesthe reduction of crotonyl-CoA to butyryl-CoA. Activity of this enzymecan be enhanced by expressing bcd in conjunction with expression of theC. acetobutylicum etfAB genes, which encode an electron transferflavoprotein. An additional candidate for the enoyl-CoA reductase stepis the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeisteret al., J. Biol. Chem. 280:4329-4338 (2005)). A construct derived fromthis sequence following the removal of its mitochondrial targetingleader sequence was cloned in E. coli resulting in an active enzyme(Hoffmeister et al., J Biol. Chem. 280:4329-4338 (2005)). This approachis well known to those skilled in the art of expressing eukaryoticgenes, particularly those with leader sequences that may target the geneproduct to a specific intracellular compartment, in prokaryoticorganisms. A close homolog of this gene, TDE0597, from the prokaryoteTreponema denticola represents a third enoyl-CoA reductase which hasbeen cloned and expressed in E. coli (Tucci and Martin, Febs Letters581:1561-1566 (2007)).

Gene name GI# Accession # Organism Bcd 15895968 NP_349317.1 Clostridiumacetobutylicum etfA 15895966 NP_349315.1 Clostridium acetobutylicum etfB15895967 NP_349316.1 Clostridium acetobutylicum TER 62287512 Q5EU90.1Euglena gracilis TDE0597 42526113 NP_971211.1 Treponema denticola

Additional enoyl-CoA reductase enzyme candidates are found in organismsthat degrade aromatic compounds. Rhodopseudomonas palustris, a modelorganism for benzoate degradation, has the enzymatic capability todegrade pimelate via beta-oxidation of pimeloyl-CoA. Adjacent genes inthe pim operon, pimC and pimD, bear sequence homology to C.acetobutylicum bcd and are predicted to encode a flavin-containingpimeloyl-CoA dehydrogenase (Harrison and Harwood, Microbiology151:727-736 (2005)). The genome of nitrogen-fixing soybean symbiontBradyrhizobium japonicum also contains a pim operon composed of geneswith high sequence similarity to pimC and pimD of R. palustris (Harrisonand Harwood, Microbiology 151:727-736 (2005)).

Gene name GI# GenBank Accession # Organism pimC 39650632 CAE29155Rhodopseudomonas palustris pimD 39650631 CAE29154 Rhodopseudomonaspalustris pimC 27356102 BAC53083 Bradyrhizobium japonicum pimD 27356101BAC53082 Bradyrhizobium japonicum

An additional candidate is 2-methyl-branched chain enoyl-CoA reductase(EC 1.3.1.52), an enzyme catalyzing the reduction of sterically hinderedtrans-enoyl-CoA substrates. This enzyme participates in branched-chainfatty acid synthesis in the nematode Ascarius suum and is capable ofreducing a variety of linear and branched chain substrates including2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA andpentanoyl-CoA (Duran et al., J Biol. Chem. 268:22391-22396 (1993)). Twoisoforms of the enzyme, encoded by genes acad1 and acad, have beencharacterized.

Gene name GI# GenBank Accession # Organism acad1 2407655 AAC48316.1Ascarius suum Acad 347404 AAA16096.1 Ascarius suum

1.4.1.a Oxidoreductase (ketone or aldehyde to amino). Oxidoreductases inthe EC class 1.4.1 that convert an aldehyde or ketone to itscorresponding amine group catalyze several biosynthetic steps in thedisclosed pathways. In FIG. 12, the conversions of OHED to 2-AHE (StepJ), 2-OHD to 2-AHD (Step H) and adipate semialdehyde to 6-aminocaproate(Step E) are catalyzed by OHED aminating oxidoreductase, 2-OHD aminatingoxidoreductase and adipate semialdehyde aminating oxidoreductase. InFIG. 13, conversion of 6-aminocaproate semialdehyde to HMD (Step H) and6-acetamidohexanal to 6-acetamidohexanamine (Step G), are also catalyzedby aminating oxidoreductases.

Most aminating oxidoreductases catalyze the reversible oxidativedeamination of alpha-amino acids with NAD+ or NADP+ as acceptor, and thereactions are typically reversible. Exemplary enzymes include glutamatedehydrogenase (deaminating), encoded by gdhA, leucine dehydrogenase(deaminating), encoded by ldh, and aspartate dehydrogenase(deaminating), encoded by nadX. The gdhA gene product from Escherichiacoli (Korber et al., J. Mol. Biol. 234:1270-1273 (1993); and McPhersonet al., Nucleic Acids Res. 11:5257-5266 (1983)), gdh from Thermotogamaritime (Kort et al., Extremophiles. 1:52-60 (1997); Lebbink et al., JMol. BioL 280:287-296 (1998); and Lebbink et al., J Mol. Biol.289:357-369 (1999)), and gdhA1 from Halobacterium salinarum (Ingoldsbyet al., Gene 349:237-244 (2005)) catalyze the reversible interconversionof glutamate to 2-oxoglutarate and ammonia, while favoring NADP(H),NAD(H), or both, respectively. The Idh gene of Bacillus cereus encodesthe LeuDH protein that has a wide of range of substrates includingleucine, isoleucine, valine, and 2-aminobutanoate (Ansorge and Kula,Biotechnol Bioeng 68:557-562 (2000); and Stoyan et al., J Biotechnol.54:77-80 (1997)). The nadX gene from Thermotoga maritime encoding forthe aspartate dehydrogenase is involved in the biosynthesis of NAD (Yanget al., J Biol. Chem. 278:8804-8808 (2003)).

Gene name GI# GenBank Accession # Organism gdhA 118547 P00370Escherichia coli Gdh 6226595 P96110.4 Thermotoga maritima gdhA1 15789827NP_279651.1 Halobacterium salinarum Ldh 61222614 P0A393 Bacillus cereusnadX 15644391 NP_229443.1 Thermotoga maritima

Lysine 6-dehydrogenase (deaminating), encoded by lysDH, catalyzes theoxidative deamination of the 6-amino group of L-lysine to form2-aminoadipate-6-semialdehyde, which in turn non-enzymatically cyclizesto form Δ¹-piperideine-6-carboxylate (Misono and Nagasaki, J. Bacteriol.150:398-401 (1982)). Exemplary enzymes can be found in Geobacillusstearothermophilus (Heydari et al., Appl Environ. Microbiol 70:937-942(2004)), Agrobacterium tumefaciens (Hashimoto et al., J Biochem.106:76-80 (1989); and Misono and Nagasaki, J. Bacteriol. 150:398-401(1982)), and Achromobacter denitrificans (Ruldeekulthamrong et al., BMB.Rep. 41:790-795 (2008)). Such enzymes are particularly good candidatesfor converting adipate semialdehyde to 6-aminocaproate given thestructural similarity between adipate semialdehyde and2-aminoadipate-6-semialdehyde.

Gene name GI# Accession # Organism lysDH 13429872 BAB39707 Geobacillusstearothermophilus lysDH 15888285 NP_353966 Agrobacterium tumefacienslysDH 74026644 AAZ94428 Achromobacter denitrificans

2.3.1.a Acyltransferase (transferring CoA to phospho). Acyltransferasesthat exchange a CoA moiety for a phosphate are in the EC class 2.3.1.Transformations in this category include the conversions of 6-AAHOP to6-acetamidohexanoyl-CoA (FIG. 13, Step K) and 6-AHOP to6-aminocaproyl-CoA (FIG. 13, Step L). Exemplary phosphate-transferringacyltransferases include phosphotransacetylase (EC 2.3.1.8), encoded bypta, and phosphotransbutyrylase (EC 2.3.1.19), encoded by ptb. The ptagene from E. coli encodes an enzyme that reversibly converts acetyl-CoAinto acetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191:559-569(1969)). This enzyme can also utilize propionyl-CoA as a substrate,forming propionate in the process (Hesslinger et al., Mol. Microbiol27:477-492 (1998)). Similarly, the ptb gene from C. acetobutylicumencodes phosphate transbutyrylase, an enzyme that reversibly convertsbutyryl-CoA into butyryl-phosphate (Walter et al., Gene 134:107-111(1993); and Wiesenborn et al., Appl Environ. Microbiol 55:317-322(1989)). Additional ptb genes are found in butyrate-producing bacteriumL2-50 (Louis et al., J. Bacteriol. 186:2099-2106 (2004)) and Bacillusmegaterium (Vazquez et al., Curr. Microbiol 42:345-349 (2001)).

Gene GenBank name GI# Accession # Organism Pta 16130232 NP_416800.1Escherichia coli Ptb 15896327 NP_349676 Clostridium acetobutylicum Ptb38425288 AAR19757.1 butyrate-producing bacterium L2-50 Ptb 10046659CAC07932.1 Bacillus megaterium

2.3.1.c Acyltransferase (N-acetyltransferase). N-Acetyltransferasestransfer an acetyl group to an amine, forming an N-acetyl group.N-Acetylation serves diverse functions in biological systems includingtranscriptional regulation, nuclear import, chromosome assembly andnucleosome remodeling (Kouzarides, EMBO J 19:1176-1179 (2000)).N-Acetylation of metabolic intermediates of arginine biosyntheticpathways serves both to protect reactive intermediates from spontaneouscyclization and also to sequester pathway intermediates from competingpathways (Caldovic and Tuchman, Biochem. J 372:279-290 (2003)).Acetylation of 6-ACA (FIG. 13, step D) serves a similar role in theproposed HMD biosynthesis route of FIG. 13, protecting reactiveintermediates from spontaneous cyclization.

One candidate enzyme for acetylating 6-ACA is lysine N-acetyltransferase(EC 2.3.1.32), an enzyme which selectively transfers the acetyl moietyfrom acetyl phosphate to the terminal amino group of L-lysine,beta-L-lysine or L-ornithine. Although this enzyme is not known toacetylate 6-ACA, this substrate is structurally similar to the naturalsubstrate. Lysine N-acetyltransferase has been characterized in Bostaurus (Paik. and Kim, Arch. Biochem. Biophys. 108:221-229, 1964) andMethanosarcina mazei (Pfluger et al., Appl Environ. Microbiol69:6047-6055 (2003)). Methanogenic archaea M. maripaludis, M.acetivorans, M. barkeri and M. jannaschii are also predicted to encodeenzymes with this functionality (Pfluger et al., Appl Environ. Microbiol69:6047-6055 (2003)).

Gene GeneBank name GI# Accession # Organism ablB 21227037 NP_632959.1Methanosarcina mazei yodP 44921183 CAF30418 Methanococcus maripaludisMA3978 20092772 NP_618847.1 Methanosarcina acetivorans MJ0635 15668816NP_247619.1 Methanocaldococcus jannaschii Mbar_A0671 73668215YP_304230.1 Methanosarcina barkeri

Alternately, 6-ACA acetylation can be catalyzed by an enzyme in the GNATfamily of N-acetyltransferases. Such enzymes transfer an acetyl groupfrom acetyl-CoA to a primary amine. The enzyme spermidineN-acetyltransferase (S SAT), also known as diamine N-acetyltransferase(EC 2.3.1.57), is capable of acetylating a variety of small moleculesubstrates. Purified enzymes from Ascaris suum and Onchocerca volvulusexhibit a broad substrate range that includes HMD (Davids et al., Mol.Biochem. Parasitol. 64:341-344 (1994); and VVittich and Walter, Mol.Biochem. Parasitol. 38:13-17 (1990)), but the associated genes have notbeen identified to date. Other enzymes with this functionality are foundin Bacillus subtilis (Forouhar et al., J. Biol. Chem. 280:40328-40336(2005)) and Homo sapiens (Casero and Pegg, FASEB J 7:653-661 (1993)). Aclosely related enzyme is thialysine N-acetyltransferase in C. elegans,an enzyme that accepts a range of substrates including lysine,ornithine, thialysine and others (bo-Dalo et al., Biochem. J 384:129-137(2004)). Amino acid residues involved in substrate binding wereidentified in the thialysine N-acetyltransferase from Leishmania major(Luersen, K., FEBS Lett. 579:5347-5352 (2005)). An additional candidateis the diaminobutyrate acetyltransferase (EC 2.3.1.178), an enzymeparticipating in ectoine biosynthesis in Methylomicrobium alcaliphilum(Reshetnikov et al., Arch. Microbiol 184:286-297 (2006)) C. salexigens(formerly Halomonas elongata) (Canovas et al., Syst. Appl Microbiol21:487-497 (1998)).

GenBank Gene name GI# Accession # Organism paiA 16080268 NP_391095.1Bacillus subtilis SSAT1 114322 P21673 Homo sapiens D2023.4 17559148NP_505978.1 Caenorhabditis elegans LmjF36.2750 68129928 CAJ09234.1Leishmania major ectA 68366269 AAY96770.1 Methylomicrobium alcaliphilum20Z ectA 6685422 Q9ZEU8.1 Chromohalobacter salexigens

An additional enzyme candidate for acetylating 6-ACA (FIG. 13, Step D)and de-acetylating 6-acetamidehexanamine (FIG. 13, Step H) is ornithineacetyltransferase (OAT, EC 2.3.1.35 and EC 2.3.1.1), a bifunctionalenzyme which catalyzes two steps of arginine biosynthesis (FIG. 14A).The first step of arginine biosynthesis (FIG. 14A, step 1) is theN-acetylation of glutamate, catalyzed by OAT with acetyl-CoA as anacetyl donor (O'Reilly and Devine, Microbiology 140 (Pt 5):1023-1025(1994)). OAT also catalyzes the fifth step of arginine biosynthesis(FIG. 14A, step 2), in which an N-acetyl group is transferred fromN-acetyl-L-ornithine to L-glutamate, the first metabolite in thearginine biosynthesis pathway. This transformation serves to recycle theacetyl group and regenerate N-acetylglutamate, conserving energy andthereby making the linear pathway a cyclic route. A similar strategy canbe employed in HMD biosynthesis from 6-aminocaproate, with a singleenzyme acetylating 6-aminocaproate and de-acetylating6-acetamidohexanamine to form HMD (FIG. 14B). Exemplary OAT enzymes areencoded by argJ in Bacillus subtilis (O'Reilly and Devine, Microbiology140 (Pt 5):1023-1025 (1994); and Sakanyan et al., Journal of GeneralMicrobiology 138:125-130 (1992)) and ECM40 in S. cerevisiae (Abadjievaet al., J Biol. Chem. 275:11361-11367 (2000); and Liu et al., Eur. JBiochem. 228:291-296 (1995)). Crystal structures of the enzymes fromyeast (Maes et al., Acta Crystallogr. Sect. F. Struct. Biol. Cryst.Commun. 62:1294-1297 (2006)) and Mycobacterium tuberculosis(Sankaranarayanan et al., Acta Crystallogr. Sect. F. Struct. Biol.Cryst. Commun. 65:173-176 (2009)) are available. Although encoded by asingle open reading frame, OAT enzymes have distinct alpha and betasubunit peptides (Liu et al., Eur. J Biochem. 228:291-296 (1995)).

Gene name GI# GenBank Accession # Organism argJ 16078185 NP_389002.1Bacillus subtilis ECM40 (ARG7) 6323707 NP_013778.1 Saccharomycescerevisiae Rv1653 15608791 NP_216169.1 Mycobacterium tuberculosis

2.3.1.d Acyltransferase (formate C-acyltransferase). The acylation ofketoacids HODH, OHED and 2-OHD to their corresponding CoA derivatives(FIG. 12, Steps L, P and Q) and concurrent release of formate, iscatalyzed by formate C-acyltransferase enzymes in the EC class 2.3.1.Enzymes in this class include pyruvate formate-lyase and ketoacidformate-lyase. Pyruvate formate-lyase (PFL, EC 2.3.1.54), encoded bypflB in E. coli, converts pyruvate into acetyl-CoA and formate. Theactive site of PFL contains a catalytically essential glycyl radicalthat is posttranslationally activated under anaerobic conditions byPFL-activating enzyme (PFL-AE, EC 1.97.1.4) encoded by pflA (Knappe etal., Proc. Natl. Acad. Sci U.S. A 81:1332-1335 (1984); and Wong et al.,Biochemistry 32:14102-14110 (1993)). A pyruvate formate-lyase fromArchaeglubus fulgidus encoded by pflD has been cloned, expressed in E.coli and characterized (Lehtio, L. and A. Goldman, Protein Eng Des Sel17:545-552 (2004)). The crystal structures of the A. fulgidus and E.coli enzymes have been resolved (Lehtio et al., J Mol. Biol. 357:221-235(2006)). Additional PFL and PFL-AE candidates are found in Clostridiumpasteurianum (Weidner and Sawers, J. Bacteriol. 178:2440-2444 (1996))and the eukaryotic alga Chlamydomonas reinhardtii (Cary et al., Appl.Environ. Microbiol 56:1576-1583 (1990)). Keto-acid formate-lyase (EC2.3.1.-), also known as 2-ketobutyrate formate-lyase (KFL) and pyruvateformate-lyase 4, is the gene product of tdcE in E. coli. This enzymecatalyzes the conversion of 2-ketobutyrate to propionyl-CoA and formateduring anaerobic threonine degradation, and can also substitute forpyruvate formate-lyase in anaerobic catabolism (Simanshu et al., JBiosci. 32:1195-1206 (2007)). The enzyme is oxygen-sensitive and, likePflB, requires post-translational modification by PFL-AE to activate aglycyl radical in the active site (Hesslinger et al., Mol. Microbiol27:477-492 (1998)).

Gene name GI# Accession # Organism pflB 16128870 NP_415423.1 Escherichiacoli pflA 16128869 NP_415422.1 Escherichia coli tdcE 48994926 AAT48170.1Escherichia coli pflD 11499044 NP_070278.1 Archaeglubus fulgidus Pfl2500058 Q46266.1 Clostridium pasteurianum Act 1072362 CAA63749.1Clostridium pasteurianum pfl1 159462978 XP_001689719.1 Chlamydomonasreinhardtii pflA1 159485246 XP_001700657.1 Chlamydomonas reinhardtii

2.6.1.a Aminotransferase. Steps E, H and J of FIG. 12 and Steps C and Gof FIG. 13 require conversion of an aldehyde or ketone to an aminogroup. This transformation can be accomplished by an aminotransferase(EC 2.6.1.-). The conversion of an aldehyde to a terminal amine (FIG.12, Step E; FIG. 13, Steps C and G) can be, catalyzed bygamma-aminobutyrate transaminase (GABA transaminase). One E. coli GABAtransaminase is encoded by gabT and transfers an amino group fromglutamate to the terminal aldehyde of succinic semialdehyde (Bartsch etal., J. Bacteriol. 172:7035-7042 (1990)). This enzyme exhibits a broadsubstrate range (Liu et al., Biochemistry 43:10896-10905 (2004)). Thegene product of puuE encodes the other 4-aminobutyrate transaminase inE. coli (Kurihara et al., J. Biol. Chem. 280:4602-4608 (2005)). GABAtransaminases in Mus musculus, Pseudomonas fluorescens, and Sus scrofahave been shown to react with 6-aminocaproic acid (Cooper, MethodsEnzymoL 113:80-82 (1985); and Scott and Jakoby, J Biol. Chem.234:932-936 (1959)).

GenBank Gene name GI# Accession # Organism gabT 16130576 NP_417148.1Escherichia coli puuE 16129263 NP_415818.1 Escherichia coli Abat37202121 NP_766549.2 Mus musculus gabT 70733692 YP_257332.1 Pseudomonasfluorescens Abat 47523600 NP_999428.1 Sus scrofa

Additional enzyme candidates include putrescine aminotransferases orother diamine aminotransferases. Such enzymes are particularly wellsuited for carrying out the conversion of 6-aminocaproate semialdehydeto HMD. The E. coli putrescine aminotransferase is encoded by the ygjGgene and the purified enzyme also was able to transaminate cadaverineand spermidine (Samsonova et al., BMC. Microbiol 3:2 (2003)). Inaddition, activity of this enzyme on 1,7-diaminoheptane and with aminoacceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) hasbeen reported (Kim, J Biol. Chem. 239:783-786 (1964); and Samsonova etal., BMC. Microbiol 3:2 (2003)). A putrescine aminotransferase withhigher activity with pyruvate as the amino acceptor thanalpha-ketoglutarate is the spuC gene of Pseudomonas aeruginosa (Lu etal., J. Bacteriol. 184:3765-3773 (2002)).

GenBank Gene name GI# Accession # Organism ygjG 145698310 NP_417544Escherichia coli spuC 9946143 AAG03688 Pseudomonas aeruginosa

Additional candidate enzymes include beta-alanine/alpha-ketoglutarateaminotransferases which produce malonic semialdehyde from beta-alanine(WO08027742). The gene product of SkPYD4 in Saccharomyces kluyveri wasshown to preferentially use beta-alanine as the amino group donor(Andersen and Hansen, Gene 124:105-109 (1993)). SkUGA1 encodes ahomologue of Saccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramoset al., Eur. J. Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes anenzyme involved in both β-alanine and GABA transamination (Andersen andHansen, Gene 124:105-109 (1993)). 3-Amino-2-methylpropionatetransaminase catalyzes the transformation from methylmalonatesemialdehyde to 3-amino-2-methylpropionate. The enzyme has beencharacterized in Rattus norvegicus and Sus scrofa and is encoded by Abat1968 (Kakimoto et al., Biochim. Biophys. Acta 156:374-380 (1968); andTamaki et al., Methods Enzymol. 324:376-389 (2000)).

GenBank Gene name GI# Accession # Organism SkyPYD4 98626772 ABF58893.1Saccharomyces kluyveri SkUGA1 98626792 ABF58894.1 Saccharomyces kluyveriUGA1 6321456 NP_011533.1 Saccharomyces cerevisiae Abat 122065191P50554.3 Rattus norvegicus Abat 120968 P80147.2 Sus scrofa

Steps J and H of FIG. 12 are catalyzed by aminotransferases thattransform amino acids into oxo-acids. In Step J, OHED is transaminatedto form 2-AHE by OHED aminotransferase. The transamination of 2-OHD to2-AHD by 2-OHD aminotransferase (Step H) is a similar reaction. Anexemplary enzyme candidate for catalyzing these reactions is aspartateaminotransferase, an enzyme that naturally transfers an oxo group fromoxaloacetate to glutamate, forming alpha-ketoglutarate and aspartate.Aspartate is similar in structure to OHED and 2-AHD. Aspartateaminotransferase activity is catalyzed by, for example, the geneproducts of aspC from Escherichia coli (Yagi et al., FEBS Lett.100:81-84, (1979); and Yagi et al., Methods Enzymol. 113:83-89 (1985)),AAT2 from Saccharomyces cerevisiae (Yagi et al., J. Biochem. 92:35-43(1982)) and ASP5 from Arabidopsis thaliana (de la Torre et al., Plant J46:414-425 (2006); Kwok and Hanson, J Exp. Bot. 55:595-604 (2004); andWilkie and Warren, Protein Expr. Purif. 12:381-389 (1998)). The enzymefrom Rattus norvegicus has been shown to transaminate alternatesubstrates such as 2-aminohexanedioic acid and 2,4-diaminobutyric acid(Recasens et al., Biochemistry 19:4583-4589 (1980)). Aminotransferasesthat work on other amino-acid substrates can catalyze thistransformation. Valine aminotransferase catalyzes the conversion ofvaline and pyruvate to 2-ketoisovalerate and alanine. The E. coli gene,avtA, encodes one such enzyme (Whalen and Berg, C. J. Bacteriol.150:739-746 (1982)). This gene product also catalyzes the transaminationof α-ketobutyrate to generate α-aminobutyrate, although the amine donorin this reaction has not been identified (Whalen and Berg, J. Bacteriol.158:571-574 (1984)). The gene product of the E. coli serC catalyzes tworeactions, phosphoserine aminotransferase and phosphohydroxythreonineaminotransferase (Lam and Winkler, J. Bacteriol. 172:6518-6528 (1990)),and activity on non-phosphorylated substrates could not be detected(Drewke et al., FEBS. Lett. 390:179-182 (1996)).

GenBank Gene name GI# Accession # Organism aspC 16128895 NP_415448.1Escherichia coli AAT2 1703040 P23542.3 Saccharomyces cerevisiae ASP520532373 P46248.2 Arabidopsis thaliana Got2 112987 P00507 Rattusnorvegicus avtA 49176374 YP_026231.1 Escherichia coli serC 16128874NP_415427.1 Escherichia coli

2.7.2.a Phosphotransferase (carboxy acceptor). Phosphotransferaseenzymes in the EC class 2.7.2 transform carboxylic acids to phosphonicacids with concurrent hydrolysis of one ATP. Steps A and E in FIG. 13require a phosphotransferase to activate the carboxyl groups of 6-ACA(Step A) and 6-acetamidohexanoate (Step E) to their correspondingphosphonic acids. Butyrate kinase carries out the reversible conversionof butyryl-phosphate to butyrate during acidogenesis in C.acetobutylicum (Cary et al., Appl. Environ. Microbiol 56:1576-1583(1990)). This enzyme is encoded by either of the two buk gene products(Huang et al., J. Mol. Microbiol Biotechnol 2:33-38 (2000)). Relatedenzyme isobutyrate kinase from Thermotoga maritima has also beenexpressed in E. coli and crystallized (Diao et al., Acta Crystallogr. D.Biol. Crystallogr. 59:1100-1102 (2003); and Diao and Hasson, J.Bacteriol. 191:2521-2529 (2009)). Aspartokinase catalyzes theATP-dependent phosphorylation of aspartate and participates in thesynthesis of several amino acids. The aspartokinase III enzyme in E.coli, encoded by lysC, has a broad substrate range and the catalyticresidues involved in substrate specificity have been elucidated (Kengand Viola, Arch. Biochem. Biophys. 335:73-81 (1996)). Two additionalkinases in E. coli are also good candidates: acetate kinase andgamma-glutamyl kinase. The E. coli acetate kinase, encoded by ackA(Skarstedt and Silverstein, J. Biol. Chem. 251:6775-6783 (1976)),phosphorylates propionate in addition to acetate (Hesslinger et al.,Mol. Microbiol 27:477-492 (1998)). The E. coli gamma-glutamyl kinase,encoded by proB (Smith et al., J. Bacteriol. 157:545-551 (1984)),phosphorylates the gamma carbonic acid group of glutamate.

GenBank Gene name GI# Accession # Organism buk1 15896326 NP_349675Clostridium acetobutylicum buk2 20137415 Q97II1 Clostridiumacetobutylicum buk2 6685256 Q9X278.1 Thermotoga maritima lysC 16131850NP_418448.1 Escherichia coli ackA 16130231 NP_416799.1 Escherichia coliproB 16128228 NP_414777.1 Escherichia coli

Acetylglutamate kinase phosphorylates acetylated glutamate duringarginine biosynthesis and is a good candidate for phosphorylating6-acetamidohexanoate (FIG. 13, Step E). This enzyme is not known toaccept alternate substrates; however, several residues of the E. colienzyme involved in substrate binding and phosphorylation have beenelucidated by site-directed mutagenesis (Marco-Martin et al., J Mol.Biol. 334:459-476 (2003); and Ramon-Maiques et al., Structure.10:329-342 (2002)). The enzyme is encoded by argB in Bacillus subtilisand E. coli (Parsot et al., Gene 68:275-283 (1988)), and ARG5,6 in S.cerevisiae (Pauwels et al., Eur. J Biochem. 270:1014-1024 (2003)). TheARG5,6 gene of S. cerevisiae encodes a polyprotein precursor that ismatured in the mitochondrial matrix to become acetylglutamate kinase andacetylglutamylphosphate reductase, an enzyme candidate for the reductionof 6-AAHOP (FIG. 13, Step F).

Gene name GI# Accession # Organism argB 145698337 NP_418394.3Escherichia coli argB 16078186 NP_389003.1 Bacillus subtilis ARG5,66320913 NP_010992.1 Saccharomyces cerevisiae

2.8.3.a Coenzyme-A transferase. Coenzyme-A (CoA) transferases catalyzethe reversible transfer of a CoA moiety from one molecule to another. InStep M of FIG. 13, 3-aminocaproyl-CoA is formed by the transfer of a CoAgroup from acetyl-CoA, succinyl-CoA, or another CoA donor. A similartransformation is catalyzed by 6-acetamidohexanoate CoA-transferase,shown in Step I of FIG. 13. Exemplary CoA transferase candidates arecatalyzed by the gene products of cat1, cat2, and cat3 of Clostridiumkluyveri which have been shown to exhibit succinyl-CoA,4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively(Seedorf et al., Proc. Natl. Acad. Sci U.S. A 105:2128-2133 (2008); andSohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). Similar CoAtransferase activities are also present in Trichomonas vaginalis (vanGrinsven et al., J Biol. Chem. 283:1411-1418 (2008)) and Trypanosomabrucei (Riviere et al., J. Biol. Chem. 279:45337-45346 (2004)).

GenBank Gene name GI# Accession # Organism cat1 729048 P38946.1Clostridium kluyveri cat2 172046066 P38942.2 Clostridium kluyveri cat3146349050 EDK35586.1 Clostridium kluyveri TVAG_395550 123975034XP_001330176 Trichomonas vaginalis G3 Tb11.02.0290 71754875 XP_828352Trypanosoma brucei

A CoA transferase that can utilize acetyl-CoA as the CoA donor isacetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit)and atoD. (beta subunit) genes (Korolev et al., Acta Crystallogr. D.Biol. Crystallogr. 58:2116-2121 (2002); and Vanderwinkel et al.,Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme has abroad substrate range (Sramek and Frerman, Arch. Biochem. Biophys.171:14-26 (1975)) and has been shown to transfer the CoA moiety toacetate from a variety of branched and linear acyl-CoA substrates,including isobutyrate (Matthies and Schink, Appl Environ. Microbiol58:1435-1439 (1992)), valerate (Vanderwinkel et al., Biochem. Biophys.Res. Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al.,Biochem. Biophys. Res. Commun. 33:902-908 (1968)). This enzyme isinduced at the transcriptional level by acetoacetate, so modification ofregulatory control may be necessary for engineering this enzyme into apathway (Pauli and Overath, Eur. J Biochem. 29:553-562 (1972)). Similarenzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al.,Appl. Environ. Microbiol 68:5186-5190 (2002)), Clostridiumacetobutylicum (Cary et al., Appl. Environ. Microbiol 56:1576-1583(1990); and Wiesenborn et al., Appl. Environ. Microbiol 55:323-329(1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al.,Biosci. Biotechnol Biochem. 71:58-68 (2007)).

Gene GenBank name GI# Accession # Organism AtoA 2492994 NP_416726Escherichia coli K12 AtoD 2492990 NP_416725 Escherichia coli K12 actA62391407 YP_226809.1 Corynebacterium glutamicum ATCC 13032 cg059262389399 YP_224801.1 Corynebacterium glutamicum ATCC 13032 ctfA 15004866NP_149326.1 Clostridium acetobutylicum ctfB 15004867 NP_149327.1Clostridium acetobutylicum ctfA 31075384 AAP42564.1 Clostridiumsaccharoperbutylacetonicum ctfB 31075385 AAP42565.1 Clostridiumsaccharoperbutylacetonicum

The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with glutaconyl-CoA and3-butenoyl-CoA (Mack et al., Eur. J. Biochem. 226:41-51 (1994)). Thegenes encoding this enzyme are gctA and gctB. This enzyme has reducedbut detectable activity with other CoA derivatives includingglutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckelet al., Eur. J Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mack et al., Eur. J. Biochem. 226:41-51(1994)).

Gene name GI# GenBank Accession # Organism gctA 559392 CAA57199.1Acidaminococcus fermentans gctB 559393 CAA57200.1 Acidaminococcusfermentans

Yet another CoA transferase is the two-unit succinyl-CoA:3:oxoacid-CoAtransferase encoded by pcaI and pcaJ in Pseudomonas putida (Kaschabek etal., J. Bacteriol. 184:207-215 (2002)). Similar enzymes based onhomology exist in Acinetobacter sp. ADP1 (Kowaichuk et al., Gene146:23-30 (1994)). Additional exemplary succinyl-CoA:3:oxoacid-CoAtransferases are present in Helicobacter pylori (Corthesy-Theulaz etal., J. Biol. Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stolset al., Protein Expr. Purif. 53:396-403 (2007)).

GeneBank Gene name GI# Accession # Organism pcaI 24985644 AAN69545.1Pseudomonas putida pcaJ 26990657 NP_746082.1 Pseudomonas putida pcaI50084858 YP_046368.1 Acinetobacter sp. ADP1 pcaJ 141776 AAC37147.1Acinetobacter sp. ADP1 pcaI 21224997 NP_630776.1 Streptomyces coelicolorpcaJ 21224996 NP_630775.1 Streptomyces coelicolor HPAG1_0676 108563101YP_627417 Helicobacter pylori HPAG1_0677 108563102 YP_627418Helicobacter pylori ScoA 16080950 NP_391778 Bacillus subtilis ScoB16080949 NP_391777 Bacillus subtilis

3.5.1.a Hydrolase (acting on linear amides). Deacetylation of linearacetamides is catalyzed by an amidohydrolase in the 3.5.1 family ofenzymes. Such an enzyme is required for the deacetylation of6-acetamidohexanamine to HMD (FIG. 13, Step H). An enzyme catalyzing asimilar transformation is 4-acetamidobutyrate deacetylase (EC 3.5.1.63),which naturally deacetylates 4-acetamidobutyrate. The enzyme, studiedfor its role in putrescine degradation in Candida boidinii (Gillyon etal., Journal of General Microbiology 133:2477-2485 (1987)), has beenshown to deacetylate a variety of substrates including6-acetamidohexanoate (Haywood and Large, Journal of General Microbiology132:7-14 (1986)). Although 6-Acetamidohexanoate is similar in structureto the desired substrate, deacetylation of this compound (FIG. 13, stepD, reverse reaction) may hinder efficient production of HMD. Proteinengineering or directed evolution may be required to improve specificityfor 6-acetamidohexanamine. The gene associated with this activity hasnot been identified to date.

2. Acetylpolyamine amidohydrolase (EC 3.5.1.62), is another candidateenzyme that forms the diamines putrescine and cadaverine from theiracetylated precursors. The acetylpolyamine deacetylase (AphA) fromMycoplana ramosa has been cloned in E. coli and characterized (Sakuradaet al., J. Bacteriol. 178:5781-5786 (1996)) and a crystal structure isavailable (Fujishiro et al., Biochem. Biophys. Res. Commun.157:1169-1174 (1988)). This enzyme has also been studied in Micrococcusluteus, but the associated gene has not been identified to date (Suzukiet al., Biochim. Biophys. Acta 882:140-142 (1986)). A protein thehistone deacetylase superfamily with high sequence similarity to AphAwas identified in the M. luteus genome (evalue=1e-18, 37% identity). TheN-acetyl-L-ornithine deacetylase from E. coli is another candidateamidohydrolase (EC 3.5.1.16). The E. coli enzyme, encoded by the argEgene (McGregor et al., J Am. Chem. Soc. 127:14100-14107 (2005); andMeinnel et al., J. Bacteriol. 174:2323-2331 (1992)), removes N-acetylgroups from a variety of substrates including ornithine, lysine,glutamine, and other amino acids (Javid-Majd and Blanchard, Biochemistry39:1285-1293 (2000)).

GenBank Gene name GI# Accession # Organism aphA 3023317 Q48935.1Mycoplana ramose MlutDRAFT_1143 172071524 EDT57566.1 Micrococcus luteusargE 16131795 NP_418392.1 Escherichia coli

4.1.1.a Carboxy-lyase. Steps D and F in FIG. 12 are catalyzed by2-ketoacid decarboxylase enzymes that generate 6-OHE and adipatesemialdehyde from OHED (Step F) and 2-OHD (Step D). In addition,alpha-ketoglutarate is decarboxylated to form pathway precursor succinicsemialdehyde by alpha-ketoglutarate decarboxylase, a keto-aciddecarboxylase. The decarboxylation of keto-acids is catalyzed by avariety of enzymes with varied substrate specificities, includingpyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chainalpha-ketoacid decarboxylase. Pyruvate decarboxylase (PDC), also termedketo-acid decarboxylase, is a key enzyme in alcoholic fermentation,catalyzing the decarboxylation of pyruvate to acetaldehyde. The enzymefrom Saccharomyces cerevisiae has a broad substrate range for aliphatic2-keto acids including 2-ketobutyrate, 2-ketovalerate, 3-hydroxypyruvateand 2-phenylpyruvate (22). This enzyme has been extensively studied,engineered for altered activity, and functionally expressed in E. coli(Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001); Li, H.and F. Jordan, Biochemistry. 38:10004-10012 (1999); and ter Schure etal., Appl. Environ. Microbiol. 64:1303-1307 (1998)). The PDC fromZymomonas mobilus, encoded by pdc, also has a broad substrate range andhas been a subject of directed engineering studies to alter the affinityfor different substrates (Siegert et al., Protein Eng Des Sel 18:345-357(2005)). The crystal structure of this enzyme is available(Killenberg-Jabs et al., Eur. J. Biochem. 268:1698-1704 (2001)). Otherwell-characterized PDC candidates include the enzymes from Acetobacterpasteurians (Chandra et al., Arch. Microbiol. 176:443-451 (2001)) andKluyveromyces lactis (Krieger et al., Eur. J. Biochem. 269:3256-3263(2002)).

Gene name GI# GeneBank Accession # Organism Pdc 118391 P06672.1Zymomonas mobilus pdc1 30923172 P06169 Saccharomyces cerevisiae Pdc20385191 Q8L388 Acetobacter pasteurians pdc1 52788279 Q12629Kluyverornyces lactis

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broadsubstrate range and has been the target of enzyme engineering studies.The enzyme from Pseudomonas putida has been extensively studied andcrystal structures of this enzyme are available (Hasson et al.,Biochemistry 37:9918-9930 (1998); and Polovnikova et al., Biochemistry42:1820-1830 (2003)). Site-directed mutagenesis of two residues in theactive site of the Pseudomonas putida enzyme altered the affinity (Km)of naturally and non-naturally occurring substrates (Siegert et al.,Protein Eng Des Sel 18:345-357 (2005)). The properties of this enzymehave been further modified by directed engineering (Lingen et al.,Protein Eng 15:585-593 (2002); and Lingen et al., Chembiochem. 4:721-726(2003)). The enzyme from Pseudomonas aeruginosa, encoded by mdlC, hasalso been characterized experimentally (Barrowman et al., FEMSMicrobiology Letters 34:57-60 (1986)). Additional gene candidates fromPseudomonas stutzeri, Pseudomonas fluorescens and other organisms can beinferred by sequence homology or identified using a growth selectionsystem developed in Pseudomonas putida (Henning et al., Appl. Environ.Microbiol. 72:7510-7517 (2006)).

Gene name GI# GenBank Accession # Organism mdlC 3915757 P20906.2Pseudornonas putida mdlC 81539678 Q9HUR2.1 Pseudomonas aeruginosa dpgB126202187 ABN80423.1 Pseudomonas stutzeri ilvB-1 70730840 YP_260581.1Pseudomonas fluorescens

A third enzyme capable of decarboxylating 2-oxoacids isalpha-ketoglutarate decarboxylase (KGI7). The substrate range of thisclass of enzymes has not been studied to date. The KDC fromMycobacterium tuberculosis (Tian et al., Proc Natl Acad Sci USA102:10670-10675 (2005)) has been cloned and functionally expressed inother internal projects at Genomatica. However, it is not an idealcandidate for strain engineering because it is large (“130 kD) andGC-rich. KDC enzyme activity has been detected in several species ofrhizobia including Bradyrhizobium japonicum and Mesorhizobium loti(Green et al., J. Bacteriol. 182:2838-2844 (2000)). Although theKDC-encoding gene(s) have not been isolated in these organisms, thegenome sequences are available and several genes in each genome areannotated as putative KDCs. A KDC from Euglena gracilis has also beencharacterized but the gene associated with this activity has not beenidentified to date (Shigeoka and Nakano, Arch. Biochem. Biophys.288:22-28 (1991)). The first twenty amino acids starting from theN-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (SEQ ID NO: 1) (Shigeokaand Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)). The gene can beidentified by testing candidate genes containing this N-terminalsequence for KDC activity.

GenBank Gene name GI# Accession # Organism Kgd 160395583 O50463.4Mycobacterium tuberculosis Kgd 27375563 NP_767092.1 Bradyrhizobiumjaponicum Kgd 13473636 NP_105204.1 Mesorhizobium loti

A fourth candidate enzyme for catalyzing this step is branched chainalpha-ketoacid decarboxylase (BCKA). This class of enzyme has been shownto act on a variety of compounds varying in chain length from 3 to 6carbons (Oku and Kaneda, J Biol Chem. 263:18386-18396 (1988); and Smitet al., Appl Environ Microbiol. 71:303-311 (2005)). The enzyme inLactococcus lactis has been characterized on a variety of branched andlinear substrates including 2-oxobutanoate, 2-oxohexanoate,2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate andisocaproate (Smit et al., Appl Environ Microbiol. 71:303-311 (2005)).The enzyme has been structurally characterized (Berg et al., Science.318:1782-1786 (2007)). Sequence alignments between the Lactococcuslactis enzyme and the pyruvate decarboxylase of Zymomonas mobilusindicate that the catalytic and substrate recognition residues arenearly identical (Siegert et al., Protein Eng Des Sel 18:345-357(2005)), so this enzyme would be a promising candidate for directedengineering Decarboxylation of alpha-ketoglutarate by a BCKA wasdetected in Bacillus subtilis; however, this activity was low (5%)relative to activity on other branched-chain substrates (Oku and Kaneda,J Biol Chem. 263:18386-18396 (1988)) and the gene encoding this enzymehas not been identified to date. Additional BCKA gene candidates can beidentified by homology to the Lactococcus lactis protein sequence. Manyof the high-scoring BLASTp hits to this enzyme are annotated asindolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvatedecarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation ofindolepyruvate to indoleacetaldehyde in plants and plant bacteria.

Gene name GI# GenBank Accession # Organism kdcA 44921617 AAS49166.1Lactococcus lactis

Recombinant branched chain alpha-keto acid decarboxylase enzymes derivedfrom the E1 subunits of the mitochondrial branched-chain keto aciddehydrogenase complex from Homo sapiens and Bos taurus have been clonedand functionally expressed in E. coli (Davie et al., J. Biol. Chem.267:16601-16606 (1992); Wynn et al., J. Biol. Chem. 267:1881-1887(1992); and Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). Inthese studies, the authors found that co-expression of chaperonins GroELand GroES enhanced the specific activity of the decarboxylase by500-fold (Wynn et al., J. Biol. Chem. 267:12400-12403 (1992)). Theseenzymes are composed of two alpha and two beta subunits.

Gene name GI# GenBankAccession # Organism BCKDHB 34101272 NP_898871.1Homo sapiens BCKDHA 11386135 NP_000700.1 Homo sapiens BCKDHB 115502434P21839 Bos taurus BCKDHA 129030 P11178 Bos taurus

The decarboxylation of 2-AHD to 6-aminocaproate (FIG. 12, Step I) iscatalyzed by an amino acid decarboxylase such as aspartatedecarboxylase. Aspartate decarboxylase participates in pantothenatebiosynthesis and is encoded by gene panD in Escherichia coli (Dusch etal., Appl. Environ. Microbiol 65:1530-1539 (1999); Merke and Nichols,FEMS Microbiol Lett. 143:247-252 (1996); Ramjee et al., Biochem. J 323(Pt 3):661-669 (1997); and Schmitzberger et al., EMBO J 22:6193-6204(2003)). Similar enzymes from Mycobacterium tuberculosis (Chopra et al.,Protein Expr. Purif. 25:533-540 (2002)) and Corynebacterium glutamicum(Dusch et al., Appl. Environ. Microbiol 65:1530-1539 (1999)) have beenexpressed and characterized in E. coli.

Gene name GI# Accession # Organism panD 67470411 P0A790 Escherichia coliK12 panD 18203593 09X4N0 Corynebacterium Glutamicum panD 54041701P65660.1 Mycobacterium tuberculosis

4.1.2.a Aldehyde-lyase. HOHD aldolase, also known as HHED aldolase,catalyzes the conversion of 4-hydroxy-2-oxo-heptane-1,7-dioate (HOHD)into pyruvate and succinic semialdehyde (FIG. 12, Step A). The enzyme isa divalent metal ion dependent class II aldolase, catalyzing the finalstep of 4-hydroxyphenylacetic acid degradation in E. coli C, E. coli W,and other organisms. In the native context, the enzyme functions in thedegradative direction. The reverse (condensation) reaction isthermodynamically unfavorable; however the equilibrium can be shiftedthrough coupling HOHD aldolase with downstream pathway enzymes that workefficiently on reaction products. Such strategies have been effectivefor shifting the equilibrium of other aldolases in the condensationdirection (Nagata et al., Appl Microbiol Biotechnol 44:432-438 (1995);and Pollard et al., Appl Environ. Microbiol 64:4093-4094 (1998)). The E.coli C enzyme, encoded by hpcH, has been extensively studied and hasrecently been crystallized (Rea et al., J Mol. Biol. 373:866-876 (2007);and Stringfellow et al., Gene 166:73-76 (1995)). The E. coli W enzyme isencoded by hpaI (Prieto et al., J. Bacteriol. 178:111-120 (1996)).

Gene name GI# GenBank Accession # Organism hpcH 633197 CAA87759.1Escherichia coli C hpal 38112625 AAR11360.1 Escherichia coli W

4.2.1.a Hydro-lyase. The enzyme OHED hydratase participates in4-hydroxyphenylacetic acid degradation, where it converts2-oxo-hept-4-ene-1,7-dioate (OHED) to 2-oxo-4-hydroxy-hepta-1,7-dioate(HODH) using magnesium as a cofactor (Burks et al., J. Am. Chem. Soc.120 (1998)) (FIG. 12, Step B). OHED hydratase enzyme candidates havebeen identified and characterized in E. coli C (Izumi et al., J Mol.Biol. 370:899-911 (2007); and Roper et al., Gene 156:47-51 (1995)) andE. coli W (Prieto et al., J. Bacteriol. 178:111-120 (1996)). Sequencecomparison reveals homologs in a range of bacteria, plants and animals.Enzymes with highly similar sequences are contained in Klebsiellapneumonia (91% identity, evalue=2e-138) and Salmonella enterica (91%identity, evalue=4e-138), among others.

Gene name GI# Gene Bank Accession # Organism hpcG 556840 CAA57202.1Escherichia coli C hpaH 757830 CAA86044.1 Escherichia coli W hpaH150958100 ABR80130.1 Klebsiella pneumoniae Sari_01896 160865156ABX21779.1 Salmonella enterica

Dehydration of 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA (FIG. 12,Step M) is catalyzed by an enzyme with enoyl-CoA hydratase activity.3-Hydroxybutyryl-CoA dehydratase (EC 4.2.1.55), also called crotonase,dehydrates 3-hydroxyisobutyryl-CoA to form crotonoyl-CoA (FIG. 14, step2). Crotonase enzymes are required for n-butanol formation in someorganisms, particularly Clostridial species, and also comprise one stepof the 3-hydroxypropionate/4-hydroxybutyrate cycle in thermoacidophilicArchaea of the genera Sulfolobus, Acidianus, and Metallosphaera.Exemplary genes encoding crotonase enzymes can be found in C.acetobutylicum (Atsumi et al., Metab Eng 10:305-311 (2008); and Boyntonet al., J. Bacteriol. 178:3015-3024 (1996)), C. kluyveri (Hillmer andGottschalk, FEBS Lett. 21:351-354 (1972)), and Metallosphaera sedula(Berg et al., Science. 318:1782-1786 (2007)) though the sequence of thelatter gene is not known.

GeneBank Gene name GI# Accession # Organism Crt 15895969 NP_349318.1Clostridium acetobutylicum crt1 153953091 YP_001393856.1 Clostridiumkluyveri

Enoyl-CoA hydratases (EC 4.2.1.17) also catalyze the dehydration of3-hydroxyacyl-CoA substrates (Agnihotri and Liu., J. Bacteriol.188:8551-8559 (2003); Conrad et al., J. Bacteriol. 118:103-111 (1974);and Roberts et al., Arch. Microbiol 117:99-108 (1978)). The enoyl-CoAhydratase of Pseudomonas putida, encoded by ech, catalyzes theconversion of 3-hydroxybutyryl-CoA to crotonoyl-CoA (Roberts et al.,Arch. Microbiol 117:99-108 (1978)). Additional enoyl-CoA hydratasecandidates are phaA and phaB, of P. putida, and paaA and paaB from P.fluorescens (Olivera et al., Proc. Natl. Acad. Sci U.S. A 95:6419-6424(1998)). The gene product of pimF in Rhodopseudomonas palustris ispredicted to encode an enoyl-CoA hydratase that participates inpimeloyl-CoA degradation (Harrison and Harwood, Microbiology 151:727-736(2005)). Lastly, a number of Escherichia coli genes have been shown todemonstrate enoyl-CoA hydratase functionality including maoC (Park andLee, J. Bacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., J.Biochem. 270:3047-3054 (2003); Park and Lee, Appl. Biochem. Biotechnol113-116:335-346 (2004); and Park and Yup, Biotechnol Bioeng 86:681-686(2004)) and paaG (Ismail et al., J. Biochem. 270:3047-3054 (2003); Parkand Lee, Appl. Biochem. Biotechnol 113-116:335-346 (2004); and Park andYup, Biotechnol Bioeng 86:681-686 (2004)).

GenBank Gene name GI# Accession # Organism Ech 26990073 NP_745498.1Pseudomonas putida paaA 26990002 NP_745427.1 Pseudomonas putida paaB26990001 NP_745426.1 Pseudomonas putida phaA 106636093 ABF82233.1Pseudomonas fluorescens phaB 106636094 ABF82234.1 Pseudomonasfluorescens pimF 39650635 CAE29158 Rhodopseudomonas palustris maoC16129348 NP_415905.1 Escherichia coli paaF 16129354 NP_415911.1Escherichia coli paaG 16129355 NP_415912.1 Escherichia coli

Alternatively, the E. coli gene products of fadA and fadB encode amultienzyme complex involved in fatty acid oxidation that exhibitsenoyl-CoA hydratase activity (Nakahigashi and Inokuchi, Nucleic AcidsRes. 18:4937 (1990); Yang, J. Bacteriol. 173:7405-7406 (1991); and Yanget al., Biochemistry 30:6788-6795 (1991)). Knocking out a negativeregulator encoded by fadR can be utilized to activate the fadB geneproduct (Sato et al., J Biosci. Bioeng 103:38-44 (2007)). The fadI andfadJ genes encode similar functions and are naturally expressed underanaerobic conditions (Campbell et al., Mol. Microbiol 47:793-805(2003)).

Gene name GI# GenBank Accession # Organism fadA 49176430 YP_026272.1Escherichia coli fadB 16131692 NP_418288.1 Escherichia coli fadI16130275 NP_416844.1 Escherichia coli fadJ 16130274 NP_416843.1Escherichia coli fadR 16129150 NP_415705.1 Escherichia coli

6.2.1.a Acid-thiol ligase (also called CoA synthetase). Steps I and M ofFIG. 13 require acid-thiol ligase or CoA synthetase functionality totransform 6-ACA and 6-acetamidohexanoate into their corresponding CoAderivatives (the terms ligase, synthetase, and synthase are used hereininterchangeably and refer to the same enzyme class). Enzymes catalyzingthese exact transformations have not been characterized to date;however, several enzymes with broad substrate specificities have beendescribed in the literature. ADP-forming acetyl-CoA synthetase (ACD, EC6.2.1.13) is an enzyme that couples the conversion of acyl-CoA esters totheir corresponding acids with the concomitant synthesis of ATP. ACD Ifrom Archaeoglobus fulgidus, encoded by AF1211, was shown to operate ona variety of linear and branched-chain substrates including isobutyrate,isopentanoate, and fumarate (Musfeldt and Schonheit, J. Bacteriol184:636-644 (2002)). A second reversible ACD in Archaeoglobus fulgidus,encoded by AF1983, was also shown to have a broad substrate range withhigh activity on cyclic compounds phenylacetate and indoleacetate(Musfeldt and Schonheit, J. Bacteriol. 184:636-644 (2002)). The enzymefrom Haloarcula marismortui (annotated as a succinyl-CoA synthetase)accepts propionate, butyrate, and branched-chain acids (isovalerate andisobutyrate) as substrates, and was shown to operate in the forward andreverse directions (Brasen and Schonheit, Arch. Microbiol 182:277-287(2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeonPyrobaculum aerophilum showed the broadest substrate range of allcharacterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferredsubstrate) and phenylacetyl-CoA (Brasen and Schonheit, Arch. Microbiol182:277-287 (2004)). Directed evolution or engineering can be used tomodify this enzyme to operate at the physiological temperature of thehost organism. The enzymes from A. fulgidus, H. marismortui and P.aerophilum have all been cloned, functionally expressed, andcharacterized in E. coli (Brasen and Schonheit, Arch. Microbiol182:277-287 (2004); and Musfeldt and Schonheit, J. Bacteriol.184:636-644 (2002)). An additional candidate is the enzyme encoded bysucCD in E. coli, which naturally catalyzes the formation ofsuccinyl-CoA from succinate with the concomitant consumption of one ATP,a reaction which is reversible in vivo (Buck et al., Biochemistry24:6245-6252 (1985)).

GenBank Gene name GI# Accession # Organism AF1211 11498810 NP_070039.1Archaeoglobus fulgidus DSM 4304 AF1983 11499565 NP_070807.1Archaeoglobus fulgidus DSM 4304 Scs 55377722 YP_135572.1 Haloarculamarismortui ATCC 43049 PAE3250 18313937 NP_560604.1 Pyrobaculumaerophilurn str. IM2 sucC 16128703 NP_415256.1 Escherichia coli sucD1786949 AAC73823.1 Escherichia coli

Another candidate enzyme for this step is 6-carboxyhexanoate-CoA ligase,also known as pimeloyl-CoA ligase (EC 6.2.1.14), which naturallyactivates pimelate to pimeloyl-CoA during biotin biosynthesis ingram-positive bacteria. The enzyme from Pseudomonas mendocina, clonedinto E. coli, was shown to accept the alternate substrates hexanedioateand nonanedioate (Binieda et al., Biochem. J 340 (Pt 3):793-801 (1999)).Other candidates are found in Bacillus subtilis (Bower et al., J.Bacteriol. 178:4122-4130 (1996)) and Lysinibacillus sphaericus (formerlyBacillus sphaericus) (Ploux et al., Biochem. J 287 (Pt 3):685-690(1992)).

Gene name GI# GenBank Accession # Organism pauA 15596214 NP_249708.1Pseudomonas mendocina bioW 50812281 NP_390902.2 Bacillus subtilis bioW115012 P22822.1 Lysinibacillus sphaericus

Additional CoA-ligases include the rat dicarboxylate-CoA ligase forwhich the sequence is yet uncharacterized (Vamecq et al., Biochem. J230:683-693 (1985)), either of the two characterized phenylacetate-CoAligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J395:147-155 (2006); and Wang et al., Biochem. Biophys. Res. Commun.360:453-458 (2007)) and the phenylacetate-CoA ligase from Pseudomonasputida (Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)).Additional candidate enzymes are acetoacetyl-CoA synthetases from Musmusculus (Hasegawa et al., Biochim. Biophys. Acta 1779:414-419 (2008))and Homo sapiens (Ohgami et al., Biochem. Pharmacol. 65:989-994 (2003))which naturally catalyze the ATP-dependant conversion of acetoacetateinto acetoacetyl-CoA.

Gene name GI# GenBank Accession # Organism Phl 77019264 CAJ15517.1Penicillium chrysogenum phlB 152002983 ABS19624.1 Penicilliumchrysogenum paaF 22711873 AAC24333.2 Pseudomonas putida AACS 21313520NP_084486.1 Mus musculus AACS 31982927 NP_076417.2 Homo sapiens

The invention will now be described in more detail on the basis of thefollowing nonlimiting examples and with reference to the accompanyingfigures.

EXAMPLES Example 1

This experiment illustrates HMD/CO₂ pH equilibrium under changingconditions. In this example, a 10% w/w aqueous HMD solution was prepared(70% HMD purchased from Sigma Aldrich). The HMD solution was heated to30° C. and initial pH recorded. Next, CO₂ (approximately 98% pure) wasbubbled into the solution for 60 min while monitoring pH. The CO₂ spargewas stopped. Air was sparged into solution for 30 min while monitoringpH. The air sparge was kept on and the solution was heated to 80° C. for40 min. The pH was measured by sampling 3 mL to a falcon tube andcooling to 30° C. The solution was heated to 88° C. for 20 min. Thesolution was cooled to 30° C. and a final pH recorded. The measured pHvalues are plotted in Graphs 1-1, 1-2 and 1-3, below. It was noted thatthe temperature probe on pH meter was reading 44.4° C. at 300 min. andcooled back down to 30° C. by the end of the 60 min. This was mostlikely caused by the exothermic acid/base reaction. This temperature,increase can also account for the nonlinearity in FIG. 27-29 because pHdecreases with increasing temperature.

TABLE 1-1 Conditions and pH response Condition change pH (temp. in ° C.)Comments Initial conditions 12.25 (30) pH probe calibrated at 22° C.Sparge CO₂ for 60 min. at 30° C.  7.96 (30) House air sparge for 30 min.at  8.23 (30) 10 bubbles/sec. sparge rate 30° C. Heated to 80° C. for 30min. w/ 10.49 (30) pH measure in a sample cooled to sparge 30° C. Heatedto 88° C. for 15 min. w/ 10.76 (30) pH measured in a sample and aftersparge 11.02 (22.5) bulk cooled down. Readings agreed +/− 0.01 Sat opento air over night 10.78 (20.6) Some decrease in pH overnight.

Table 1-2 shows simulated molar ratio of CO₂:HMD based on pH. It can beobserved that 1.8 equivalents of CO₂ go into solution very quickly. Therate of absorption starts to slow down significantly when 2 equivalentsare reached.

TABLE 1-2 HMD:CO₂ in solution for selected points based on pHsimulation. Simulated CO₂:HMD pH (molar ratio in solution) 10.76 0.5239.77 1.314 9.00 1.820 8.23 1.994 7.96 2.038 12.433 Control for = 0.86M,pKw = 13.840

The results illustrate that air and heat can restore the pH all the wayback up to 11.02. Extraction should be possible at this pH. Aerationduring fermentation may be able to strip off some of the dissolved CO₂.

Example 2

This example reports the results of a simulated fermentation process.Briefly, CO₂ (approximately 98% pure) and HMD (70% HMD from SigmaAldrich) were fed into an MM9 solution that had the followingcomposition:

-   -   0.68% (6.8 g/L) Disodium phosphate Na₂HPO₄    -   0.3% (3 g/L) Monopotassium phosphate KH₂PO₄    -   0.15%(1.5 g/L) Ammonium chloride NH₄C1    -   0.1% (1 g/L) Ammonium sulfate (NH₄)₂SO₄    -   0.05% (0.5 g/L) Sodium chloride NaCl

The experimental conditions for this example are listed in Table 2-1.

TABLE 2-1 Summary of Experimental conditions Input Number Unit Feed rateHMD 0.6 mL/min Feed [HMD] 1.16 mol/L Initial reactor volume 0.6 L Totaltime of exp. 24 hours Final solution volume 1.464 L Final [HMD] 0.685mol/L Molar ratio CO₂ 3.2 to 1 HMD Temp. of inlet CO₂ 21 ° C. CO₂ feedrate 54.8 mL/min Air to CO₂ ratio 4 to 1 CO₂ Air feed rate 219 mL/minTotal gas sparge rate 274 mL/min

The measured pH of the solution and concentration of HMD added over timeare set out in FIG. 30.

This feed rate used in this example corresponds to an average rate of3.31 g/L/hr (a reasonable production rate in an industrial fermentationprocess). The final titer and feed concentration of HMD was confirmed byLCMS. The HMD and CO₂ reach a dynamic equilibrium around pH 8.5. As moreHMD is added, more CO₂ is absorbed. The simulated HMD:CO₂ ratio is 1.95for pH=8.53. This ratio indicates that the major species in solution isthe HMD²⁺(HCO₃)₂ salt (hexamethylenediamine bis-bicarbonate). FIGS. 31and 32 illustrate the relative concentration of species as a function ofpH for HMD and H₂CO₃, respectively.

At pH 8.5 (dashed, black line) the major species in solution are thediprotonated HMD and bicarbonate. A minority presence of HMD-carbamatemay be present as well.

Example 3

This example demonstrates the generation of HMD, as the free base, fromthe protonated and/or carbonate/carbamate compounds formed by contactingan aqueous solution of HMD with CO₂ gas, where the aqueous solution wasMM9 medium.

A 250 mL four-neck flask was fitted With a condenser, temperature probe,and air sparge needle. The system was open to air through the top of thecondenser. The feed was an aqueous HMD solution in MM9 where pH wasadjusted to 8.68 with CO2. The feed solution was analyzed for HMDconcentration by LCMS ad the pH was measured. The solution was spargedwith air and refluxed at 85° C. for three hours. The resultingsolution's pH was taken and the concentration of HMD measured by LCMS.

TABLE 3-1 Summary of regeneration results [HDM] Stream pH mo1/L CO₂:HMDratio* Feed  8.68 @ 834.0 1.91 21.7° C. Product 11.26 @ 954.7 0.27723.5° C. *Based on calculation from pH.

The positive pH change from 8.68 to 11.26 shown in Table 3-1 indicatesthat CO₂ was stripped out of solution. The concentration increasedslightly as some water escaped through the condenser and the solutionwas concentrated. The fraction of HMD in free base form at pH 11.26 is57%.

Example 4

The example describes the solvent extraction of HMD from the CO₂stripped solution prepared in Example 3 above as well as extracting HMDfrom an aqueous solution without pH adjustment as a control for solventperformance. The following protocol was used in this example:

The pH of the aqueous feed was measured. In a 50 mL falcon tube, 20 g ofsolvent was mixed with 20 g of feed. This combination was furtheraggressively mixed in the falcon tube for 5 minutes and vortexed for 1minute. The tube with the mixed solvent and feed was allowed to settleuntil phase separation was complete. The volume of the bottom, aqueouslayer was recorded. A sample of the top layer was carefully pipetted outthe top layer. A sample the bottom layer was also obtained and the pH ofthe bottom layer measured. The recovery, distribution coefficients andselectivity based on mass balance were calculated.

The extraction data for three solvents extracting HMD from water are setout in Table 4-1.

TABLE 4-1 Results of solvent screening. HMD in deionized (DI) water.[HMD] = 835 mM Solvent % pH pH % used* Solubility before after % waterin extract Selectivity recovery 1-hexanol 0.59 12.70 12.31 11.68 10.7666.4 Isopentanol 2.8 12.70 12.16 9.08 19.07 71.4 Cyclohexanol 3.6 12.7012.36 16.65 8.13 70.9 Hexane 9.4 × 10⁻⁴ 12.55 12.54 <0.001 >1,000 9.1*All solvents were received from Sigma Aldrich at >97% purity

Based on the screening reported in Table 4-1, 1-hexanol was initiallyused for extracting the product of the HMD regeneration solutionprepared in Example 3 above because it has the lower water solubilitythan isopentanol and cyclohexanol. Alkanes, specifically hexane, werescreened and subsequently tested due to extremely low water solubility.Hexane extracted little if any water and provided reasonable recovery ofthe available free base.

Table 4-2 illustrates that with 1:1 solvent to feed ratio, HMD may beextracted in 25.8% overall recovery or 4% in the case of hexane. Basedon the pH before extraction, only 60% of free base HMD is available.Thus, approximately 43% of the available free base HMD was extracted bythe solvent 1-hexanol and about 7% by hexane. Hexane extracted little ifany water and provided reasonable recovery of the available free base.

TABLE 4-2 Results of extracting stripped solution from “regenerationexperiment.” [HMD] = 955 mM % % Solvent % pH pH water in re- usedSolubility before after extract Selectivity covery 1-hexanol 0.59 11.4411.02 9.02 2.70 25.8 Hexane 9.4 × 10−4 11.48 11.42 <0.001 >1,000 4.0

Example 5 Comparative Example

When a modeled HMD fermentation was controlled to a pH 7 with H2SO4,assuming 88% of glucose is converted to HMD on a mass basis, and a finalHMD titer of 116 g/L, 0.843 g H2SO4 per g HMD is needed to maintain thepH at 7. Due to the amount of sulfuric acid used, carbon dioxide is notreadily absorbed. This resulted in a final DIC/TDCA value of less than0.5%. The fermentation model takes into account, among other things,cellular growth and respiration, byproduct formation, and mediacomposition needed.

Example 5A

When an HMD fermentation was modeled at a pH of 8.5, assuming 88% ofglucose is converted to HMD on a mass basis, and a final HMD titer of116 g/L, no sulfuric acid is needed to maintain the pH of 8.5 (based onexperimental results). Carbon dioxide is readily absorbed by the HMD.During the seed fermentation, the DIC/TDCA rose from <1% toapproximately 54%. During the product fermentation, the value rose fromapproximately 54% to approximately 96%. The fermentation model takesinto account, among other things, cellular growth and respiration,byproduct formation, and media composition needed.

Example 5B

When a modeled HMD fermentation is controlled to a pH of 7 with onlyCO2, assuming 88% of glucose is converted to HMD on a mass basis, and afinal HMD titer of 116 g/L, It is shown that 2.4 moles of CO2 would needto be absorbed per mole of HMD. It has been experimentally shown inother examples that a pH of 7 can be reached with CO2 as the only acidused in pH control. During the seed fermentation model, the DIC/TDCArose from <1% to approximately 82%. During the product fermentationmodel, the value rose from approximately 82% to approximately 97%. Thefermentation model takes into account, among other things, cellulargrowth and respiration, byproduct formation, and media compositionneeded.

Example 6

Solvents such as alkanes were evaluated as suitable solvents for HMDfree base recovery from aqueous solutions. See FIGS. 33 and 34. ASPEN(Aspen Plus ver 8.6; Aspen Technology, Inc., USA) software was used tomodel percent HMD recovered from a 50% aqueous solution of HMD bysolvent extraction with hexane or heptane. The components included inthe Aspen model were water, HMD, DIC (dissolved inorganic carbon), andsolvent (hexane or heptane). An electrolyte NRTL model (ENRTL-RK) inASPEN was used. A 50% HMD solution was modeled since that is anattainable concentration, especially using the methods described hereinfor water and CO2 removal, and is believed to be a concentration thatavoids or reduces precipitation of HMD. For the model CO2 content in the50% HMD solution was 0.3%. However, as the efficiency of solventextraction increases with higher HMD concentrations, the HMDconcentration limit for solvent extraction may be even higher.

In this example the extraction column had 10 theoretical stages. In thismodel the HMD comprised about 96.6% free base (solvent extractableform), thus the plotted values slightly underestimate the percentrecovery of the recoverable form of HMD. These in silico modelingresults demonstrate that alkanes can be effective solvents for HMDrecovery from aqueous solutions at a range of solvent to HMD solutionratios.

The efficiency of solvent extraction increases with decrease in DICconcentration as shown below, supporting the importance of CO2 removal.DIC decrease results in higher pH and higher concentration ofrecoverable free base form.

Example 7

ASPEN Plus was used to model the use of either a water evaporator (amulti-effect evaporator) or a steam stripping column alone to achievewater and CO2 removal prior to HMD recovery. Conditions were as above;the components included in the ASPEN model were water, HMD, DIC speciesand hexane; an electrolyte NRTL model was used. While either step can beremoved, and despite increasing electricity and steam usage in theevaporator when more water is evaporated, a savings in utility costs isrealized with use of the evaporator because it is more efficient atremoving water than the stripping column. The figure below (FIG. 35)shows steam and electricity usages as a function of total water removedwhen no stripping column is present. The plotted utility costs are forthe evaporator step. The maximum (“Max”) steam and electricity usagerefer to the point where a 50% HMD by weight aqueous solution wasachieved. As mentioned above this HMD concentration was selected for themodel as it is believed suitable for extraction, avoiding further waterand CO₂ removal which could lead to precipitate formation as thesolution is concentrated. As the efficiency of solvent extractionincreases with higher HMD concentrations, further concentration may beuseful.

The figure below (FIG. 36) demonstrates steam usage in a strippingcolumn in the case where no evaporator is present. A concentration limitis a 50% by weight HMD solution. As more water is evaporated from thefeed, the duty and steam usage of the reboiler increases significantly.However, this is partially offset by the lower capital cost of astripping column compared to an evaporation unit. The “Max” point on theFIG. 36 indicates the point where the 50% HMD solution is obtained.

Example 8

Preparation of an HMDA Producing Microbiol Organism Having CarbonicAnhydrase

Escherichia coli is used as a target organism to engineer to produceHMDA having nucleic acids encoding the enzymes utilized in the HMDApathway and carbonic anhydrase.

The gene encoding a Desulfovibrio vulgaris (GenBank accession ACL09337.1GI:218758438, SEQ ID) is codon-optimized for expression in E. coli andis cloned into an expression vector under the control of a constitutivepromoter. This vector also contains an origin of replication and anantibiotic resistance gene. Also cloned into an expression vector orintegrated into the host, E. coli in this example, are genes encodingenzymes for production of a diamine, e.g, HMD.

The resulting plasmids are transformed into E. coli, for example MG1655or ATCC 8739, by chemical transformation or electroporation. Forchemical transformation, cells are grown to mid-log growth phase, asdetermined by the optical density at 600 nm (0.5-0.8). The cells areharvested, washed and finally treated with CaCl2). To chemicallytransform these E. coli cells, purified plasmid DNA is allowed to mixwith the cell suspension in a microcentrifuge tube on ice. A heat shockis applied to the mixture and followed by a 30-60 minute recoveryincubation in rich culture medium. For electroporation, E. coli cellsgrown to mid-log growth phase are washed with water several times andfinally resuspended into 10% glycerol solution. To electroporate DNAinto these cells, a mixture of cells and DNA is pipetted into adisposable plastic cuvette containing electrodes. A short electric pulseis then applied to the cells to form small holes in the membrane whereDNA could enter. The cell suspension is then incubated with rich liquidmedium followed by plating on solid agar plates. Detailed protocol isdescribed in Molecular Cloning: A Laboratory Manual Third Edition,Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press, 3rdEdition.

The resulting genetically engineered E. coli is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). The expression of thecarbonic anhydrase and HMDA genes are corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to producecarbonic anhydrase can be confirmed using phenolphtheleine pH indicatorto monitor pH change as carbonic anhydrase converts carbonate to CO₂ andthe resulting pH of the solution containing the DA increases. The colorchange of phenolphtheleine can be monitored by absorbance at 550 nm. Anassay is conducted by adding 300 mM-1000 mM DA to a cell lysate or theextracellular media with 300 mM-400 mM KHCO₃ and 400 μM-1100 μMphenolphthalein (Alvizo, et. al. 2014 PNAS 111(46): 16436-16441).Another assay for carbonic anhydrase activity is a colorimetric assayusing 4-nitrophenylacetate as a substrate; 3 mM 4-nitrophenylacetate(Verpoorte et. al. 1967 J. Biol. Chem. 242: 4221-4229. Carbonicanhydrase can alas be monitored by production of CO2. HMD can beconfirmed by HPLC.

What is claimed:
 1. A process for diamine (DA) production comprising thesteps of: a) culturing a genetically engineered microorganism comprisinga diamine (DA) synthesis pathway selected from the group consisting ofhexamethylenediamine, cadaverine, putrescine, ethylenediamine andheptamethylenediamine with at least one exogenous nucleic acid encodingat least one enzyme of the DA synthesis pathway for producing a DA inmedium under suitable conditions and for a sufficient period of time toproduce DA and form one or more of DA carbonate, DA bicarbonate, and/orDA bis-bicarbonate in a cultured medium and a carbonic anhydrase insufficient amount to (a) enhance the formation of the DA carbonate, DAbicarbonate, and/or, DA bis-bicarbonate by converting carbon dioxide toa bicarbonate and/or carbonate ions, (b) enhance the release of carbondioxide from a solution of DA carbonate, DA bicarbonate, and/or DAbis-bicarbonate by converting a bicarbonate and/or carbonate ions tocarbon dioxide, or (c) both (a) and (b); wherein i) carbon dioxide,carbonate, bicarbonate or carbonic acid predominantly control pH of themedium; ii) percent dissolved inorganic carbon (DIC) in the medium isgreater than or equal to 40%; and the DIC is determined by the formula:DIC/TDCA×100 where TDCA is the Total Dissolved Counter Anions and is thesum of DIC and other anions; or iii) at least 40% of diamine species inthe medium comprises one or more of DA carbonate, DA bicarbonate, and/orDA bis-bicarbonate; b) converting at least one or more of the DAcarbonate, DA bicarbonate, and/or DA bis-bicarbonate into DA free baseand carbon dioxide; and c) isolating the DA free base.
 2. The process ofclaim 1, further comprising removing solids from the cultured medium toform the DA carbonate, DA bicarbonate, and/or DA bis-bicarbonate.
 3. Theprocess of claim 1, further comprising removing water from the DA freebase mixture and optional recycling water to a fermenter.
 4. The processof claim 1, further comprising extracting the DA free base with organicsolvent in an extractor to form an extracted DA free base solution andaqueous raffinate, and optionally recycling the aqueous raffinate to theextractor.
 5. The process of claim 1, further comprising distilling theDA free base to form purified DA free base and organic solvent,optionally recycling the solvent to the extractor, and optionallyremoving undesired impurities.
 6. The process of claim 1, furthercomprising adding an aqueous base to remove the DA carbonate, DAbicarbonate, and/or DA bis-bicarbonate from the DA free base.
 7. Theprocess of claim 1, wherein ≥50% to ≤99% of the diamine in the mediumcomprises one or more of a DA carbonate, DA bicarbonate, and/or DAbis-bicarbonate.
 8. The process of claim 1, wherein the geneticallyengineered microorganism further forms one or more of carbon dioxide,carbonate, bicarbonate or carbonic acid.
 9. The process claim 1, whereinone or more of carbon dioxide, carbonate, bicarbonate or carbonic acidis externally added to the medium.
 10. The process claim 1, wherein theconverting at least one or more of the DA carbonate, DA bicarbonate,and/or DA bis-bicarbonate into DA free base and carbon dioxide is byheating.